Methods and apparatus for conducting multiple measurements on a sample

ABSTRACT

Multiplexed test measurements are conducted using an assay module having a plurality of assay domains. In preferred embodiments, these measurements are conducted in assay modules having integrated electrodes with a reader apparatus adapted to receive assay modules, induce luminescence, preferably electrode induced luminescence, in the wells or assay regions of the assay modules and measure the induced luminescence.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of copending application Ser.No. 12/977,271, filed Dec. 23, 2010, which is a divisional of copendingapplication Ser. No. 10/238,391, filed Sep. 10, 2002, now U.S. Pat. No.7,858,321, which claims priority to U.S. Provisional Application No.60/363,498, filed Mar. 11, 2002; U.S. Provisional Application Ser. No.60/318,293, filed Sep. 10, 2001; U.S. Provisional Application No.60/318,284, filed Sep. 10, 2001; and U.S. Provisional Application Ser.No. 60/318,289, filed Sep. 10, 2001, each of which are herebyincorporated by reference.

FIELD OF THE INVENTION

This application relates to reagents, apparatus, systems, kits andmethods for conducting multiple chemical, biochemical and/or biologicalassays on a sample.

BACKGROUND OF THE INVENTION

At this time, there are a number of commercially available instrumentsthat utilize electrochemiluminescence (ECL) for analytical measurementsincluding drug screening. Species that can be induced to emit ECL(ECL-active species) have been used as ECL labels. Examples of ECLlabels include: i) organometallic compounds where the metal is from, forexample, the noble metals of group VIII, including Ru-containing andOs-containing organometallic compounds such as thetris-bipyridyl-ruthenium (RuBpy) moiety and ii) luminol and relatedcompounds. Species that participate with the ECL label in the ECLprocess are referred to herein as ECL coreactants. Commonly usedcoreactants include tertiary amines (e.g., see U.S. Pat. No. 5,846,485),oxalate, and persulfate for ECL from RuBpy and hydrogen peroxide for ECLfrom luminol (see, e.g., U.S. Pat. No. 5,240,863). The light generatedby ECL labels can be used as a reporter signal in diagnostic procedures(Bard et al., U.S. Pat. No. 5,238,808, herein incorporated byreference). For instance, an ECL label can be covalently coupled to abinding agent such as an antibody, nucleic acid probe, receptor orligand; the participation of the binding reagent in a bindinginteraction can be monitored by measuring ECL emitted from the ECLlabel. Alternatively, the ECL signal from an ECL-active compound may beindicative of the chemical environment (see, e.g., U.S. Pat. No.5,641,623 which describes ECL assays that monitor the formation ordestruction of ECL coreactants). For more background on ECL, ECL labels,ECL assays and instrumentation for conducting ECL assays see U.S. Pat.Nos. 5,093,268; 5,147,806; 5,324,457; 5,591,581; 5,597,910; 5,641,623;5,643,713; 5,679,519; 5,705,402; 5,846,485; 5,866,434; 5,786,141;5,731,147; 6,066,448; 6,136,268; 5,776,672; 5,308,754; 5,240,863;6,207,369; 6,214,552 and 5,589,136 and Published PCT Nos. WO99/63347;WO00/03233; WO99/58962; WO99/32662; WO99/14599; WO98/12539; WO97/36931and WO98/57154.

Commercially available ECL instruments have demonstrated exceptionalperformance. They have become widely used for reasons including theirexcellent sensitivity, dynamic range, precision, and tolerance ofcomplex sample matrices. The commercially available instrumentation usesflow cell-based designs with permanent reusable flow cells. Recently,ECL instrumentation has been disclosed that uses reagents immobilized onthe electrode used to induce ECL (see, e.g., U.S. Pat. Nos. 6,140,045;6,066,448; 6,090,545; 6,207,369 and Published PCT Appl. No. WO98/12539).Multi-well plates having integrated electrodes suitable for such ECLmeasurements have also been recently disclosed (see, e.g., copendingU.S. application Ser. Nos. 10/185,274 and 10/185,363 (entitled “AssayPlates, Reader Systems and Methods for Luminescence Test Measurements”,each filed on Jun. 28, 2002, hereby incorporated by reference). Thesemulti-well plates having integrated electrodes include plates havingmultiple assay domains within a well.

The use of multi-well assay plates allows for the parallel processingand analysis of multiple samples distributed in multiple wells of aplate. Typically, samples and reagents are stored, processed and/oranalyzed in multi-well assay plates (also known as microplates ormicrotiter plates). Multi-well assay plates can take a variety of forms,sizes and shapes. For convenience, some standards have appeared for someinstrumentation used to process samples for high throughput assays.Assays carried out in standardized plate formats can take advantage ofreadily available equipment for storing and moving these plates as wellas readily available equipment for rapidly dispensing liquids in and outof the plates. Some well established multi-well plate formats includethose found on 96-well plates (12×8 array of wells), 384-well plates(24×16 array of wells) and 1536-well plate (48×32 array of well). TheSociety for Biomolecular Screening has published recommended microplatespecifications for a variety of plate formats, the recommendedspecifications hereby incorporated by reference.

SUMMARY OF THE INVENTION

The present invention includes apparatus, systems, system components,reagents, kits and methods for performing a plurality of assays on asample. The invention includes assay modules having one or more assaycells (e.g., wells, compartments, chambers, channels, flow cells, etc.)that comprise a plurality of assay domains (e.g., discrete locations ona module surface where an assay reaction occurs and/or where an assaysignal is emitted for carrying out a plurality of assay measurements.The assay cell is, preferably, adapted to hold a volume of fluid incontact with assay domains within the assay cell without contactingassay domains in other assay cells of an assay module. In preferredembodiments, the assay modules are multi-well plates, the platescomprising a plurality of wells, one or more of the wells comprising aplurality of assay domains (referred to herein as Multi-DomainMulti-Well Plates or MDMW Plates). Preferably, the plates are designedto be compatible with plate handling equipment (e.g., fluid dispensers,plate washers, plate stackers, plate movers, and/or plate readers)designed for use with standard format multi-well plates.

The assays of the invention are preferably coupled to a detection stepthat involves the use of an electrode, the generation of light, and themeasurement of the generated light. Examples of processes that may beused in such a detection step include electrochemiluminescence (alsoreferred to as electrogenerated chemiluminescence), electroluminescence,and chemiluminescence triggered by an electrochemically- generatedspecies. For the purposes of the application and for convenience, thesethree processes will be referred to as “electrode induced luminescence”.Electrochemiluminescence involves electrogenerated species and theemission of light. For example, electrochemiluminescence may involveluminescence generated by a process in which one or more reactants aregenerated electrochemically and undergo one or more chemical reactionsto produce species that emits light, preferably repeatedly. Theinvention also relates to assays and measurements that do not requirethe use of an electrode, for example, the assays of the invention may bebased on measurements of chemiluminescence, fluorescence,bioluminescence, phosphorescence, optical density and processes thatinvolve the emission of light from a scintillant. The invention alsorelates to assays and measurements that do not involve luminescence, forexample, the assays of the invention may be based on measurements ofelectrochemical processes (e.g., processes involving the measurement orgeneration of current or voltage), electrical processes (e.g., processesinvolving the measurement of resistance or impedance), surface plasmonresonance or optical interference effects.

Accordingly, in certain preferred embodiments of the invention, theassay modules and/or MDMW Plates are adapted to allow assay measurementsto be conducted using electrode induced luminescence measurements (mostpreferably, electrochemiluminescence measurements), e.g., as describedin copending U.S. application Ser. Nos. 10/185,274 and 10/185,363(entitled “Assay Plates, Reader Systems and Methods for LuminescenceTest Measurements”), each filed on Jun. 28, 2002, hereby incorporated byreference. Advantageously, assay domains patterned on a surface of awell (e.g., on an electrode in a well adapted for conducting electrodeinduced luminescence measurements) are defined by physical boundarieswhich can include ledges or depressions in the surface, patternedmaterials deposited or printed on the surface, and or interfaces betweenregions of the surface that vary in a physical property (e.g.,wettability). Such physical boundaries simplify the patterning ofreagents on surfaces of a well by confining and preventing the spreadingof small drops of reagents applied to an assay domain.

By providing two levels of multiplexing (multiple wells per plate andmultiple domains per well), MDMW Plates provide a variety of advantagesover conventional multi-well plates that only have one assay domain perwell. For example, a MDMW Plate having N wells and M assay domains perwell allows a panel of M assays to be run on a plurality of N samples.Conducting the same series of assays on conventional N-well plates wouldrequire M plates, M times more sample and reagents, and considerablymore pipetting and plate handling steps to achieve the same performance.Conducting the same series of assays on conventional array “chips” wouldinvolve the handling and movement of N chips and would likely not becompatible with standard plate handling equipment designed for use withmulti-well plates. Conducting the same series of assays on a singleultra-high density multi-well plate with M×N wells would generally leadto reduced assay sensitivity (sample volume and, therefore, number ofanalyte molecules, tends to scale inversely with the density of wells ona plate) as well as to other problems associated with ultra-high densityplate formats (e.g., expensive and complicated fluid dispensingequipment, lack of mixing, evaporative losses, trapping of air bubbles,inability to carry out wash steps, etc.).

The invention includes assay formats that take advantage of multiplexingavailable through the use of assay cells comprising multiple assaydomains and, in particular, through the use of MDMW Plates. Someexamples of preferred assay formats are described below. It isunderstood that while some of the forms are described in terms of MDMWPlates, they can be applied to other assay modules comprising assaycells with multiple domains. It is also understood that the multiplicityof assay domains available within an assay cell allows many of theformats described below to be combined within one cell.

In one preferred assay format, a plurality of analytes or activities aremeasured within one well of a MDMW plate. For example, panels of assaysmay be developed for measuring a plurality of analytes or activitiesassociated with a particular biological system (e.g., panels ofimmunoassays or hybridization assays for monitoring cytokine mRNA orprotein levels), disease state (e.g., panels of assays for cardiacmarkers, for identifying allergens responsible for allergic reactions,for identifying infectious organisms, etc.), tissue type, organism,class of protein, enzyme or biological molecule, etc. In one embodiment,a panel of assays is used to provide a fingerprint for identifying abiological system (e.g., a pattern of analyte levels associated with aparticular cell type, organelle type, organism type, tissue type,bacteria or virus). For example, a plurality of assays for differentcomponents found within a genus of biological systems can be used toidentify species or subspecies within that genus. In another embodiment,a differential measurement involving a plurality of assays for differentcomponents within a biological system is used to identify the state ofthe biological system (e.g., diseased vs. normal state, activated vs.normal state, etc.) or to identify the components within a biologicalsystem that are affected by an external condition or stimulus (e.g.,changes in the distribution of components associated with development ofa disease state, addition of a stimulatory species, addition of apotential drug candidate, changes in environmental conditions such aspH, temperature, etc.). Assay panels may also be used to determine thefunction of one or more proteins. For example, a protein may be screenedagainst a patterned library of enzyme substrates and/or potentialbinding partners to identify enzymatic or binding activities.Conversely, a patterned library of proteins may be exposed to a knownbiological material to determine if any of the proteins binds to, reactswith or is otherwise transformed by the biological material.

In another preferred assay format, some fraction of the assay domainspresent in a well are devoted to internal standards, controls orcalibrators. For example, one or more assay domains may be left uncoatedor may be coated with a blocking agent or a biomaterial not expected toparticipate in a reaction with a sample; such assay domains may be usedto measure and/or correct for non-specific binding of labels to surfacein the well. In another example, one or more assay domains is coatedwith a labeled reagent (e.g., a reagent labeled with an ECL label); suchassay domains may be used to measure and/or correct for conditions thatmay affect the generation and measurement of signal (e.g., ECL) from alabel (e.g., pH, temperature, chemical interferents, colored species,etc.). In another example, one or more assay domains are used to carryout a control assay for a control analyte that is spiked into the assaymixture. Preferably, the control assay is similar in format to assayscarried out on other assay domains. Control assays may be used tomeasure and/or correct for non-specific binding, conditions that affectthe generation of signal from a label and conditions that affect assayreactions (variations in incubation time, temperature, mixing, etc.).

In another preferred assay format the same analyte or activity ismeasured in multiple domains within a well; such redundancy can allowfor greater statistical confidence in an assay result. Such multiplemeasurements of the same analyte may involve the use of multiple roughlyidentical assay domains or, alternatively, may involve the use of assaydomains that vary in some property (for example, domain size, domainlocation, surface density of an assay reagent, blocking agent, assayreagent affinity, assay reagent specificity, assay format, sensitivityto interferents, sensitivity to temperature, assay kinetics, sensitivityto optical distortion, etc.) so as to account for, detect, and/orcompensate for a source of assay error (e.g., inconsistent ornon-homogenous mixing, steric crowding of assay reagents in an assaydomain, non-specific binding, matrix effects, interfering species,imprecise temperature control, imprecise timing of assay steps,exceeding of assay dynamic range, variation in fluid volume or meniscusshape, etc.).

In another preferred assay format, the same analyte or activity ismeasured in multiple domains within a well, the domains being comprisedon individually addressable electrodes. In such a system one may measurethe kinetics of an assay reaction by sequentially applying electricalenergy to individual assay domains at selected times and measuring thechange in electrical current, electrical potential, or, preferably,electrode induced luminescence (most preferably, ECL) over time. Bymeasuring different time points at different assay domains, it is notnecessary to repeatedly apply possibly damaging electrical energy to thesame assay domain.

In another preferred assay format, the same analyte is measured atdifferent assay domains within a well, the different assay domains beingdesigned to measure a different property or activity of the analyte. Inone embodiment, an enzyme with multiple different activities is measuredin a well comprising different assay domains that differ in theirselectivity for each enzymatic activity of the enzyme (e.g., assaydomains that comprise substrates for selected enzymatic activitiesand/or assay domains that are capable of capturing and measuring thesubstrates or products of selected enzymatic activity), that aredesigned to measure binding activities of the enzyme (e.g., assaydomains comprising potential binding partners of the enzyme or that aredesigned to capture the enzyme so as to allow the measurement ofinteraction with potential binding partners in solution) and/or assaydomains designed to measure the ability of the enzyme to act as asubstrate for a second enzyme (e.g., binding domains designed to allowfor a specific binding assay of the product of the action of the secondenzyme on the first enzyme). In another embodiment, a well comprises adomain for measuring the amount of an enzyme (e.g., via a binding assaysuch as an immunoassay) and one or more other domains for measuring oneor more activities associated with the enzyme; this embodiment allowsthe measured activity to be referenced to the amount of enzyme. Theinclusion of assay domains capable of capturing an enzyme of interesthas the added advantage of allowing the purification of the enzyme froma crude sample within the assay well. In yet another embodiment of theinvention, a well comprises an assay domain capable of capturing anenzyme of interest and one or more additional assay domains formeasuring an activity of the enzyme of interest. Methods using such awell may include a wash step for purifying the enzyme from impurities ina crude enzyme preparation.

In another preferred assay format, the number of measurements that canbe carried out in one well is increased by co-immobilizing a pluralityof assay reagents in each domain within the well. For example, one canscreen for the binding partner of a biomaterial of interest bypatterning a library of M potential binding partners on M assay domainsin a well, exposing the well to a sample containing a labeledbiomaterial and looking for the assay domain that produces a signalindicative of a binding event. Alternatively, one can pattern a libraryof M×I potential binding partners by co-immobilizing I potential bindingpartners in each assay domain. A signal at a specific assay domain wouldindicate that one of I potential binding partners has binding activity;the identity of the binding partner could be determined by thenindividually testing each component of that assay domain.Advantageously, an assay kit may contain a first MDMW plate thatmultiplexes assay reagents within assay domains of a well and a set ofadditional MDMW plates that are patterned so as to allow the testing ofeach individual component of an assay domain in the first plate (e.g., asecond MDMW Plate having a well with a plurality of assay domains eachcomprising one component of an assay domain on the first plate).

In another preferred embodiment, the number of assay components that canbe patterned and uniquely identified on an array of M domains(where M isan integer greater than three) is increased by patterning each reagentinto a unique group of assay domains. For example, one can pattern alibrary of up to Z=(M!)/[2!(M−2)!] potential binding partners(preferably, >M binding partners) so that up to (M−1) binding partnersare immobilized in each domain but one or more (preferably, all) bindingpartners are immobilized in a unique pair of domains (the other bindingpartners, preferably, being immobilized in unique sets of one domain. Inthis case, those one or more binding partners can be identified bylooking for pairs of assay domains producing signals indicative of abinding event. By way of example, wells comprising 4, 7, 10, and 25assay domains have, respectively, 6, 21, 45 and 300 unique pairs ofdomains per well. Similarly, one can pattern a library of up to(M!)/[Z!(M−Z)!] potential binding partners (preferably, >M bindingpartners) so that one or more (preferably, all) binding partners areimmobilized in a unique group of Z domains. The number of componentsthat can be screened in a given well can be further increased bypatterning some components in groups of Z₁ domains, others in groups ofZ₂ domains and so on, where Z₁, Z₂, . . . are integers greater than orequal to one and less than or equal to M.

In another preferred assay format, potentially cross-reacting analytesare measured in different domains in the same well. For example, twosimilar analytes (a first analyte and a second analyte) may be measuredusing two assay domains comprising binding reagents (a first domainselective for the first analyte and a second domain selective for thesecond analyte) even if the binding reagents are only partiallyselective for each analyte. By carrying out the assays in the same well,the binding of the second analyte to the second domain reduces itseffective concentration in solution and reduces its ability to interferewith the measurement of the first analyte at the first domain. To theextent that such effects do not completely eliminate cross-reactions,the ability to measure both cross-reacting species allows for themathematical deconvolution of signals so as to further reduce the effectof cross-reactions on assay results. Such deconvolutions can, e.g., bebased on empirical calibrations (e.g., using a two dimensional matrix ofcalibrators varying the concentrations of both analytes, preferably thecalibrators are chosen and the results modeled using Design ofExperiment techniques) or on theoretical models (e.g., models derivedusing the thermodynamic and/or kinetic parameters associated with eachpossible binding interaction). Optionally, only the first analyte ismeasured and the second domain serves only to sequester the secondanalyte and prevent it from interfering with the measurement of thefirst analyte. The methods described above for reducing cross-reactionsand interferences can be used to i) reduce and/or account forinterfering substances in crude biological samples (e.g., blood, plasma,serum, tissue extracts, cell extracts) such as bilirubin, lipid,hemaglobin and/or ii) to aid in preventing and/or to prevent assayinterference and cross-reactions from closely related species, e.g., toaid in measuring and distinguishing between closely related drugs andrelated metabolites, steroidal hormones and related metabolites,vitamins and related metabolites, modified forms of proteins, nucleicacids and saccharides (e.g., different phosphorylation states of ananalyte, between different degradation states of an analyte, differentbound states of an analyte, etc.), etc.

In another preferred assay format, a plurality of different assaydomains in a well are adapted to measure different forms of an analyteof interest. By way of example, domains may be adapted to measure freeand bound forms of an analyte of interest (e.g., free PSA vs. boundPSA), to measure unmodified and/or modified forms of an analyte ofinterest (examples of modifications that can be measured include, butare not limited to, phosphorylation, ubiquitnation, prenylation,myristoylation, glyosidation), and/or to measure cleavage or degradationproducts of an analyte (e.g., protease, nuclease or glycosidaseproducts). Alternatively, one assay domain generically measures thetotal amount of multiple forms of an analyte and a second assay domainis specific for one form of the analyte (e.g., for measuring free PSAvs. total PSA).

In another preferred assay format, the starting material/substrate andproduct (and, optionally, intermediates and/or side products) in areaction of interest are measured at different assay domains of a wellof a MDMW Plate. In one embodiment, measurements in different wells arecarried out for different reaction times, allowing for a completekinetic characterization of the reaction. In a second embodiment, assaysfor the starting material and product (or any two species producedand/or consumed in the reaction) show some level of cross-reactivity; asdescribed above, measurement of both species can be used to reduce theeffect of the cross-reactivity. In a third embodiment, measurement ofboth starting material and product allows one to correct for variationsin the original amount of the starting material. Such corrections areespecially important when following reactions or activities in complexbiological systems such as cells or tissue. For example, in followingthe phosphorylation of a cellular receptor in response to activation ofthe cell, it is desirable to correct the measured amount ofphosphorylated receptor to account for variations in the level ofreceptor protein expression in the cell line. Measurement ofphosphorylated and nonphosphorylated forms of the receptor allows theextent of phosphorylation to be expressed as a percentage.Alternatively, the same information can be obtained through measurementsof total receptor and phosphorylated receptor.

In another preferred assay format, a well comprises a first assay domaincontaining a labeled substrate for a cleavage reaction and a secondassay domain containing a binding reagent capable of capturing theproduct of the cleavage reaction. Preferably, the substrate is linked toa label (preferably an ECL label) such that the cleavage reactionresults in the release, from the first assay domain of a cleavageproduct linked to the label. The extent of the cleavage reaction may befollowed by measuring the drop of signal from the first assay domain andthe increase in signal from the second assay domain. By way of example,the binding reagent may be an antibody (e.g., an antibody specific for apeptide released by proteolytic activity) or a nucleic acid probe (e.g.,a probe specific against an oligonucleotide released by a nucleaseactivity) directed against the cleavage product. Alternatively, thesubstrate may be further linked to a capture moiety (e.g., a hapten orbiotin) such that the released cleavage product comprises both a labeland a capture moiety. In this alternate embodiment, the binding reagentcan be a binding reagent directed against the capture moiety (e.g., anantibody directed against the hapten, avidin, or streptavidin).

In another preferred assay format, a library of enzymes isco-immobilized with binding reagents capable of capturing enzymeproducts so as to form an array of assay domains having assay domainsthat contain both an enzyme and a binding reagent capable of capturing aproduct of the enzyme. Such an array allows the signals derived from theenzyme reactions to be produced in a pattern that corresponds to thearrangement of enzymes. In one embodiment, the enzymes are paired withbinding reagents that are preferentially specific for the product ofthat enzyme. In another embodiment, the binding reagents are capable ofbinding the products of a plurality of enzymes in the well. In such acase, the assay domains are spaced appropriately and carried out underappropriate conditions (e.g., in the absence of mixing) to increase theprobability that a product produced in one domain will bind bindingreagents in that same domain before it has the opportunity to diffuse toa different domain. For example, a library of tyrosine kinases may bepatterned into an array of assay domains, each domain also comprising ananti-phosphotyrosine antibody. Introduction of one or more tyrosinekinase substrates (preferably, linked to a label, most preferably,linked to an ECL label) leads to phosphorylation of the substrates andcapture of the labeled product by the anti-phosphotyrosine antibody. Thelabeled product produced in a domain will be preferentially captured byantibodies in the same domain, ensuring that the signal generated in adomain is representative of the activity of the enzyme in that domain.

In another preferred assay format, multiple assay domains are used toaid in screening antibodies (or other binding reagents) for a bindingreagent with a desired specificity for a binding species. Samples (e.g.,supernatants from hybridoma cultures) are contacted with a plurality ofassay domains. One assay domain comprises the binding species. Theothers include controls for specificity and cross-reactivity (e.g.,closely related substances, potential assay interferents, a carrierprotein used in an immunization procedure used to generate the bindingreagents, linkers used in generating carrier protein-hapten conjugates,etc.). In one embodiment, the binding of a binding reagent can bedetected using a labeled secondary binding reagent that broadly binds aclass of binding reagents (e.g., an anti-species antibody). In anotherembodiment, a plurality of binding domains comprise a panel ofanti-species antibodies directed against different antibody classes(alternatively, any antibody class specific binding reagent may be used)and the binding domains are contacted with a hybridoma supernatant (orother sample containing antibodies) in order to determine the class ofthe antibody in the supernatant. In one preferred embodiment, thebinding of binding reagents to specific domains is detected using alabeled secondary binding reagent that broadly binds a class of bindingreagents (e.g., an anti-species antibody) so as to measure the amount ofall antibodies in the sample. Alternatively, a labeled hapten may beused as the detection reagent so that only the class of antibodieshaving a desired specificity is determined.

In another preferred assay format, multiple assay domains are used toexpand the dynamic range of an assay beyond what can be achieved using asingle assay domain. For example, a binding assay may involve the use ofa plurality of binding domains comprising binding reagents that differin their affinity for the analyte of interest. The domain with thehighest affinity binding reagent is used to measure low concentrationsof analyte. Domains with intermediate or weak affinity binding reagentsare used for samples having intermediate or high concentrations ofanalyte. In the case of assay domains with intermediate or weak affinitybinding reagents, the binding reagents are, preferably, selected to havedissociation constants roughly centered in the range of analyteconcentrations to be measured by that assay domain.

In the specific case of sandwich binding assays that experience a hookeffect at high concentrations, the dynamic range of the assay may beexpanded by pairing the sandwich immunoassay (conducted in a first assaydomain) with a competitive assay for the same analyte (conducted in asecond assay domain). Preferably, the competitive assay involves thecompetition of analyte in a sample with an immobilized analog of theanalyte for binding to a labeled anti-analyte antibody. More preferably,the analog of the analyte does not comprise the epitope recognized bythe capture antibody in the sandwich assay (e.g., the analog of theanalyte may be a peptide fragment derived from a protein analyte thatdoes not include the epitope recognized by the capture antibody). Thesandwich and competitive assays may use the same labeled detectionantibody. Advantageously, the amount of detection antibody is roughlyequal to the sum of the amount of analog of the analyte and captureantibody. For amounts of analyte lower than the amount of captureantibody, the sandwich assay will give a signal roughly linearlydependent on the concentration of analyte and the competitive assay willbe roughly independent of analyte concentration. Amounts of analytehigher than the amount of capture antibody will lead to decreases issignal from both the sandwich assay (due to hook effect, i.e., theincrease in the probability that the analyte will be bound to only oneof the detection or capture antibody and not both at the same time) andthe competitive assay (due to competition). In this region, thecompetitive assay may be used to quantify analyte or to simply warn thatthe dynamic range of the sandwich assay has been exceeded. In analternate embodiment, capture reagents are chosen that differ in bindingkinetics; the binding time and kinetic constants are chosen so that i)low concentrations of analyte are measured in fast binding assay domainsand ii) high concentrations of analyte (exceeding the binding capacityof the assay domains) are measured in kinetically controlled bindingreactions at slow binding domains.

In another preferred assay format, one or more assay domains in a wellare used for purposes other than as a solid phase for a solid phaseassay. By way of example: i) an assay domain may comprise bindingreagents for sequestering an assay interferent; ii) an assay domain maycomprise binding reagents for capturing and purifying a biologicalmaterial (e.g., a protein of unknown function, an enzyme, an enzymesubstrate, a binding partner in a binding reaction, etc.) from a crudepreparation such as blood, serum, cell lysates, tissue samples, etc.and/or iii) an assay domain may be used as a location for storing driedreagents to be rehydrated and dissolved during the course of an assay(e.g., binding reagents, enzymes, enzyme substrates, controls,calibrators, buffers, blocking agents, detergents, labeled-reagents, ECLcoreactants, inhibitors, drug candidates, etc.). Preferably, the driedreagents in one assay domain are prevented, during preparation andstorage of an assay well, from contacting the other assay domains in awell so as to prevent unwanted interactions between reagents. In suchcase, the dried reagents do not contact other assay domains untilsufficient sample volume is added to the well to spread the sampleacross all the domains Advantageously, when storing dried reagents in anassay domain, the assay domain is surrounded by a physical boundary(e.g., ledges or depressions on a surface of the well, patternedmaterials deposited or printed on the surface, and or interfaces betweenregions of the surface that vary in a physical property such aswettability) that allows small drops of fluids to be confined on theassay domain but also allows a larger volume of fluid to spread overmultiple domains. In one embodiment of a competitive binding assay, oneassay domain of a well comprises an immobilized binding reagent andanother assay domain comprises a dried labeled competitor; thisarrangement prevents the competitor from binding to the binding reagentprior to the addition of sample. In one embodiment of a sandwich bindingassay, a first assay domain of a well comprises an immobilized capturebinding reagent and a second assay domain comprises a dried labeledbinding reagent; this arrangement prevents the labeled binding reagentfrom binding non-specifically to the first assay domain, e.g., duringdrying or storage of the reagents. In one embodiment of an enzymeinhibition assay, a first assay domain of a well comprises an enzymesubstrate (dried on or immobilized in the assay domain) and a secondassay domain comprises a dried enzyme; this arrangement prevents theenzyme from acting on the substrate prior to the addition of the samplecontaining an inhibitor.

The invention includes assay modules and MDMW Plates adapted to carryout assays using one or more assay formats of the invention, methods ofusing the modules or plates, methods of making the modules or plates,kits including the plates and one or more reagents used in an assay, andsystems including plates and apparatuses for reading plates. Theinvention includes the measurement of analytes or chemical, biologicalor biochemical activities using the modules, plates or methods of theinvention. The invention also includes the measurement or identification(e.g., in a screen of a library of potential drugs) of modulators (e g ,inhibitors or enhancers) of such chemical, biological or biochemicalactivities. The invention also includes the application of the plates,modules or methods of the invention to the characterization of aprotein. For example a protein may be screened against a library ofbiological materials to identify biological materials that bind theprotein, accept the protein as an enzymatic substrate, are modified byan enzymatic activity of the protein, or otherwise interact with theprotein. Conversely, a biological material may be screened against alibrary of proteins to identify the proteins that bind the biologicalmaterial, accept the biological material as an enzymatic substrate, aremodified by an activity of the biological material, or otherwiseinteract with the biological material.

DESCRIPTION OF THE FIGURES

FIG. 1A is a schematic representation, according to one embodiment ofthe invention, of a panel of binding assays.

FIG. 1B is a schematic representation, according to one embodiment ofthe invention, of a panel of sandwich binding assays.

FIG. 1C is a schematic representation, according to one embodiment ofthe invention, of a panel of competitive binding assays.

FIG. 1D is a schematic representation, according to one embodiment ofthe invention, of a panel of enzyme assays.

FIG. 2 is a schematic representation, according to one embodiment of theinvention, of an assay panel that includes a test binding assay, acontrol for non-specific binding, a control for the efficiency of signalgeneration and transmission, and a control binding assay.

FIG. 3 is a schematic representation, according to one embodiment of theinvention, of an assay panel that includes assays for several activitiesof an enzyme with a plurality of activities.

FIG. 4 is a schematic representation, according to one embodiment of theinvention, of an assay panel comprising a binding assay for an enzymeand a binding assay for an enzyme product.

FIG. 5 is a schematic representation, according to one embodiment of theinvention, of an assay panel comprising binding assays for the substrateand product of an enzymatic reaction.

FIG. 6 is a schematic representation, according to one embodiment of theinvention, of an assay panel for a cleaving enzyme comprising an assaydomain having a labeled substrate for the enzyme and an assay domaincomprising a binding reagent capable of capturing a labeled product ofthe invention.

FIG. 7 is a schematic representation, according to one embodiment of theinvention, of an assay panel comprising an array of assay domainscomprising different enzymes, the enzymes being co-immobilized forbinding reagents capable of binding enzymatic products.

FIGS. 8A-8C are schematic representations of expanded dynamic rangebinding assays, according to preferred embodiments of the invention,comprising three assay domains of varying affinity for the analyte.

FIGS. 9A-9B are schematic representations of expanded dynamic rangebinding assays, according to preferred embodiments of the invention,comprising a sandwich binding assay and a competitive binding assay forthe same analyte.

FIG. 10A shows a layered view of MDMW plate 1000, a MDMW plate that isadapted for electrode induced chemiluminescence measurements.

FIG. 10B shows a stylized cross sectional view of 3 wells of MDMW plate1000, a MDMW plate that is adapted for electrode inducedchemiluminescence measurements.

FIG. 10C shows dielectric layer 1140, a modification of dielectric layer1040 shown in FIGS. 10A and 10B.

FIG. 10D shows a stylized cross-sectional view of 3 wells of MDMW plate1100, a modification of plate 1000 employing dielectric layer 1140

FIG. 11 shows a view of a MDMW plate adapted for electrode inducedchemiluminescence measurements.

FIG. 12 is a schematic description of an assay for two activities of HIVRT enzyme.

FIGS. 13A and 13B are graphical representations of the inhibition of twoactivities of HIV RT by an inhibitor.

FIG. 14 is a graphical representation of the selectivity of a MDMW Platedesigned to measure 4 different infectious agents.

FIG. 15 is a table showing the selectivity of a MDMW Plate designed tomeasure two different kinase activities.

FIG. 16 plots signal as a function of the concentration of bovine IgGthat is labeled with biotin and a sulfonated derivative of Ru(bpy)₃.Data is plotted for MDMW Plates having avidin-coated assay domains thatvary in number and size.

FIGS. 17A-17D demonstrate the independent measurement by ECL sandwichimmunoassay of four analytes (IL-1β, IL-6, TNF-α and IFN-γ) in wells ofa multi-well assay plate. The working electrode in each well ispatterned with four assay domains, each assay domain comprising acapture antibody specific for one of the analytes. The plots show theECL signal emitted from each assay domain as a function of theconcentration of each analyte.

FIG. 18 is a CCD camera image showing the independent measurement by ECLsandwich immunoassay of four analytes (IL-1β, IL-6, TNF-α and IFN-γ) inwells of a multi-well assay plate. The working electrode in each well ispatterned with four assay domains, each assay domain comprising acapture antibody specific for one of the analytes. The figure shows animage of the ECL emitted from a sector of wells used to assay samplescontaining varying mixtures of the four analytes. The highlighted wellis annotated to show the arrangement of the four assay domains. Thatspecific well was used to assay a sample having 250 pg/mL each of IL-1βand TNF-α and 8 pg/mL each of IL-6 and IFN-γ.

FIG. 19A is a schematical representation of a 4-spot well adapted for anassay for EGF induced Receptor Autophosphorylation at Tyrosine 1173using MSD™ Standard 4-spot MULTI-ARRAY® Plates according to oneembodiment of the invention. FIGS. 19B-19D are CCD images of wells ofthe plate having different concentrations of EGF.

DETAILED DESCRIPTION OF THE INVENTION

The assay domains of the invention may be adapted to carry out assays ina wide range of formats. Preferably, assay measurements are coupled tothe capture or release of detectable label (e.g., an enzyme, particle,photoluminescent species, chemiluminescent species,electrochemiluminescent species, electroactive species, radioactivespecies, magnetic species, etc.) from a solid phase, preferably, asurface of an assay domain. Preferably, the label is detectable byelectrode induced luminescence (most preferably,electrochemiluminescence) and the solid phase is an electrode adapted toinduce electrode induced luminescence (preferably,electrochemiluminescence). By analogy, the assay concepts describedherein can also be applied to solid phase assay formats that do notrequire the use of a label such as surface plasmon resonance and opticalinterference techniques.

FIGS. 1A-1D are schematic representations that show selected examples ofassay panels that may be carried out in assay cells (preferably, wellsof a MDMW Plate) comprising multiple assay domains. FIG. 1A illustratesa panel of binding assays carried out in a well 100 of a MDMW Platehaving assay domains 105A-C, comprising immobilized binding reagents110A-C directed against labeled analytes 115A-C. Suitable bindingreagent/analyte pairs are known in the art and include antibody/hapten,antibody/antigen, receptor/ligand, nucleic acid sequence/complementarysequence, lectin/sugar, nucleic acid/nucleic acid binding protein,protein/protein (e.g., proteins that dimerize, aggregate form bindingcomplexes), etc.

FIG. 1B illustrates a panel of sandwich binding assays carried out in awell 120 of a MDMW Plate having assay domains 125A-C comprisingimmobilized capture binding reagents 130A-C and soluble detectionbinding reagents 132A-C directed against analytes 135A-C. In onepreferred embodiment, the panel is a panel of sandwich immunoassays. Inanother preferred embodiment, the panel is a panel of sandwich nucleicacid hybridization assays.

FIG. 1C illustrates a panel of competitive binding assays carried out ina well 140 of a MDMW Plate having assay domains 145A-C. Analytes 155A-Ccompete with competitors 152A-C for binding to binding reagents 150A-C.If the competitor of an analyte is labeled, the corresponding bindingreagent is immobilized, or visa versa. In one preferred embodiment, thepanel is a panel of competitive immunoassays.

FIG. 1D illustrates a panel of enzyme assays carried out in well 160 ofa MDMW Plate. Enzyme 170A cleaves labeled substrate 175 (immobilized inassay domain 165A) to release a labeled product from the assay domain.Enzyme 170B joins substrate 176 (immobilized in assay domain 165B) andlabeled substrate 177 to link the label to the assay domain. Enzyme 170Cmodifies substrate 178 (immobilized in assay domain 165C) to make aproduct 179 that is recognized by labeled binding reagent 180. Enzyme170D catalyzes the conversion of labeled substrate 181 to labeledproduct 182. Labeled product 182 is then captured by binding reagent 183(immobilized in assay domain 165D). In an alternate embodiment, thelabel is omitted from substrate 181 and product 182 and product 182 isdetected by addition of a labeled detection binding reagent to form asandwich complex. Enzymes (and other chemical, biochemical, and/orbiological activities) that can be measured by one or all of the formatsdescribed in FIG. 1D include, but are not limited to, nucleic acidpolymerases, nucleic acid ligases, helicases, integrases, nucleases,proteases, protein synthesis, glycosidases, phosphatases, kinases,prenylation enzymes, myristoylation enzymes, etc.

Useful panels include panels of assays for analytes or activitiesassociated with a specific biochemical system, biochemical pathway,tissue, organism, cell type, organelle, disease state, class ofreceptors, class of enzymes, etc. Preferred panels include immunoassayfor cytokines and/or their receptors (e.g., one or more of TNF-α, TNF-β,IL1-α, IL1-β, IL2, IL4, IL6, IL10, IL12, IFN-γ, etc.), growth factorsand/or their receptors (e.g., one or more of EGF, VGF, TGF, VEGF, etc.),second messengers (e.g., cAMP, cGMP, phosphorylated forms of inositoland phosphatidyl inositol, etc.) drugs of abuse, therapeutic drugs,auto-antibodies (e.g., one or more antibodies directed against the Sm,RNP, SS-A, SS-B Jo-1, and Scl-70 antigens), allergen specificantibodies, tumor markers, cardiac markers (e.g., one or more ofTroponin T, Troponin I, myoglobin, CKMB, etc.), markers associated withhemostasis (e.g., one or more of Fibrin monomer, D-dimer,thrombin-antithrombin complex, prothrombin fragments 1 & 2, anti-FactorXa, etc.), markers of acute viral hepatitis infection (e.g., one or moreof IgM antibody to hepatitis A virus, IgM antibody to hepatitis B coreantigen, hepatitis B surface antigen, antibody to hepatitis C virus,etc.), markers of Alzheimers Disease (β-amyloid, tau-protein, etc.),markers of osteoporosis (e.g., one or more of cross-linked N orC-telopeptides, total deoxypyridinoline, free deoxypyridinoline,osteocalcin, alkaline phosphatase, C-terminal propeptide of type Icollagen, bone-specific alkaline phosphatase, etc.), markers offertility (e.g., one or more of Estradiol, progesterone, folliclestimulating hormone (FSH), luetenizing hormone (LH), prolactin, β-hCG,testosterone, etc.), markers of congestive heart failure (e.g., one ormore of β-natriuretic protein (BNP), α-natriuretic protein (ANP),endothelin, aldosterone, etc.), markers of thyroid disorders (e.g., oneor more of thyroid stimulating hormone (TSH), Total T3, Free T3, TotalT4, Free T4, and reverse T3), and markers of prostrate cancer (e.g., oneor more of total PSA, free PSA, complexed PSA, prostatic acidphosphatase, creatine kinase, etc.). Preferred panels also includenucleic acid arrays for measuring mRNA levels of mRNA coding forcytokines, growth factors, components of the apoptosis pathway,expression of the P450 enzymes, expression of tumor related genes, etc.Preferred panels also include nucleic acid arrays for genotypingindividuals (e.g., SNP analysis), pathogens, tumor cells, etc. Preferredpanels also include libraries of enzymes and/or enzyme substrates (e.g.,substrates and/or enzymes associated with ubiquitination, proteaseactivity, kinase activity, phosphatase activity, nucleic acid processingactivity, GTPase activity, guanine nucleotide exchange activity, GTPaseactivating activity, etc.). Preferred panels also include libraries ofreceptors or ligands (e.g., panels of G-protein coupled receptors,tyrosine kinase receptors, nuclear hormone receptors, cell adhesionmolecules (integrins, VCAM, CD4, CD8), major histocompatibility complexproteins, nicotinic receptors, etc.). Preferred panels also includelibraries of cells, cell membranes, membrane fragments, reconstitutedmembranes, organelles, etc. from different sources (e.g., from differentcell types, cell lines, tissues, organisms, activation states, etc.).

Applications of panels include the determination of a state of abiological system, the detection or identification of disease state. Thedetermination of analytes associated with a state of a biological system(e.g., by differential measurements of a plurality of analytes insamples derived from normal or diseased biological systems or fromnormal and activated biological systems, etc.). Panels may also beemployed in drug screening. Through the use of panels, the effect of apotential drug on a plurality of biological activities (e.g., bindinginteractions or enzymatic activities) can be determined in one well of aMDMW Plate. Panels may also be used to speed up the characterization ofa protein. For example a protein may be screened against a library ofbiological materials to identify biological materials that bind theprotein, accept the protein as an enzymatic substrate, are modified byan enzymatic activity of the protein, or otherwise interact with theprotein. Conversely, a biological material may be screened against alibrary of proteins to identify the proteins that bind the biologicalmaterial, accept the biological material as an enzymatic substrate, aremodified by an activity of the biological material, or otherwiseinteract with the biological material.

Some assay domains in an assay cell or well may be reserved for assaycontrols or calibrators. FIG. 2 is a schematic representation, accordingto one embodiment of the invention, of an assay panel that includes atest binding assay for an analyte of interest, a control fornon-specific binding, a control for the efficiency of signal generationand transmission, and a control binding assay. FIG. 2 shows a well 200of a MDMW Plate having assay domains 210A-D. Assay domain 210A comprisesa capture binding reagent 215 (e.g., an antibody or a nucleic acid)specific for the analyte of interest 217. Assay domain 210B comprises ablocking agent 225 (e.g., BSA, or bovine IgG) that, preferably, was alsoused to block open sites in assay domain 210A. Alternatively, blockingagent 225 is a reagent with similar properties to capture bindingreagent 215 except that it is not expected to interact with samplesintroduced into the assay. Assay domain 210C comprises a labeled reagent235. Assay domain 210D comprises a capture binding reagent 245 specificfor control analyte 247. The well also comprises labeled detectionantibody 219 that is specific for analyte 215, labeled detectionantibody 249 that is specific for control analyte 247, an unknownquantity of analyte 217, and a predetermined amount of control analyte247. Formation of a sandwich complex in assay domain 210A allows formeasurement of the analyte. Assay domain 210B is used to measure theamount of background signal including non-specific binding of thelabeled reagents. Assay domain 210C is used to control for factors thatinfluence the efficiency of signal generation by the label and theefficiency of signal detection. Measurement of a sandwich complex inassay domain 210D is used to control for factors that influence theefficiency of binding reactions. In alternate embodiments, assay domains210A and/or 210D comprise reagents for conducting other types ofmeasurements such as other binding assay formats or enzymatic activityassays.

FIG. 3 illustrates an assay, according to one embodiment of theinvention, for the activities of an enzyme with multiple activities. Theassay is carried out in well 300 of a MDMW Plate. Enzyme 370 cleaveslabeled substrate 375 (immobilized in assay domain 365A) to release alabeled product from the assay domain. Enzyme 370 also joins substrate376 (immobilized in assay domain 365B) and labeled substrate 377 to linkthe label to the assay domain. Enzyme 370 also modifies substrate 378(immobilized in assay domain 365C) to make a product 379 that isrecognized by labeled binding reagent 380. Enzyme 370 also catalyzes theconversion of labeled substrate 381 to labeled product 382. Labeledproduct 382 is then captured by binding reagent 383 (immobilized inassay domain 365D). In an alternate embodiment, the label is omittedfrom substrate 381 and product 382 is detected by addition of a labeleddetection binding reagent to form a sandwich complex.

FIG. 4 illustrates an assay, according to one embodiment of theinvention, of an enzymatic activity. Well 400 of a MDMW Plate comprisesassay domain 410 having an immobilized capture binding reagent 412(e.g., an antibody) capable of binding enzyme 415 and assay domain 420comprising an immobilized binding reagent 425 (e.g., an antibody)capable of binding a product of enzyme 415. Addition of a samplecontaining the enzyme leads to the capture of the enzyme in assay domain420. Optionally, a wash step may be introduced to remove interferingsubstances in the enzyme sample. Addition of a labeled substrate resultsin the generation of a labeled product that is captured and measured inassay domain 420. Addition of labeled detection reagent 417 (e.g., anantibody) allows for the measurement of the amount of enzyme 415 inassay domain 410 via sandwich binding assay. This measurement allows themeasured enzymatic activity to be referenced to the amount of enzyme.Alternatively, enzyme 410 is labeled and labeled detection reagent 417may be omitted. In another alternative embodiment, labeled detectionreagent 417 is omitted and enzyme 415 is captured and, optionally,purified but not directly measured. In a preferred embodiment of theassay, enzyme 415 is a phosphatase or kinase and binding reagent 425 isan antibody that preferentially binds to one of a phospho-peptide or itsnonphosphorylated form.

FIG. 5 illustrates an assay, according to one embodiment of theinvention, where an enzymatic activity is measured by measuring theconsumption of substrate and the generation of product. Well 500 in aMDMW Plate comprises an assay domain 510A comprising immobilized capturebinding reagent 512 specific for labeled enzyme substrate 515 and anassay domain 510B comprising immobilized capture binding reagent 517specific for labeled enzyme product 520. A sample comprising a mixtureof labeled substrate 515 and labeled product 520 resulting from theaction of an enzyme on the enzyme substrate is introduced into well 500.Measurement of substrate and product by binding assay allows the extentof conversion to be calculated even if the initial amount of substratewas unknown. Alternatively, substrate 515 is not labeled and substrate515 and product 520 are measured via sandwich binding assay orcompetitive binding assay. In a preferred embodiment of the invention,the enzyme is a kinase or phosphatase and the binding reagents areantibodies specific for the phosphorylated or non-phosphorylated form ofa peptide or protein. Alternatively, one capture reagent is specific foreither product or substrate and the other capture reagent binds bothequally. This panel allows the measurement of product or substrate inone domain and the combined total of product and substrate in the otherdomain.

FIG. 6 illustrates an assay, according to one embodiment of theinvention, for an enzyme with a cleaving activity. Well 600 of a MDMWPlate comprises assay domain 610 comprising an immobilized labeledsubstrate 615 and assay domain 620 comprising an immobilized bindingreagent 622 that is specific for labeled enzyme product 625. Enzyme 630cleaves substrate 615 forming product 625 which is captured in assaydomain 620. The assay format allows the measurement of both theconsumption of substrate and the production of product. Optionally,substrate 615 is not labeled and product 625 is measured via a sandwichor competitive binding assay. In a preferred embodiment, enzyme 630 is aprotease, substrate 615 is a labeled peptide, and binding reagent 622 isan antibody specific for the peptide. Alternatively, substrate 615 alsocomprises a capture moiety and binding reagent 622 is specific for thecapture moiety.

FIG. 7 illustrates an assay format, according to one embodiment of theinvention, for measuring the activity of an immobilized array ofenzymes. Well 700 of a MDMW Plate comprises assay domains 710A-C whichcomprise immobilized enzymes 715A-C and immobilized binding reagents720A-C, the binding reagents being specific for a product of the enzymeco-immobilized in the same assay domain. Introduction of labeledsubstrates 725A-C (which may the same or different) leads to thegeneration of labeled products 730A-C (which may be the same ordifferent). The close proximity of the enzymes to binding reagents leadsto preferential capture of products in the assay domain in which theywere produced (as opposed to the diffusion and capture of products inadjacent domains). Preferably, the assay domains are in slightdepressions in the bottom of the well so as to inhibit convection nearthe surface of the domains and to inhibit horizontal diffusion away fromthe surface of the domains. In an alternate embodiment, the substratesare not labeled and the product is measured via a sandwich orcompetitive binding assay. In one preferred embodiment, the enzymes arekinases, the substrates have consensus sequences for specific members ofthe kinase library, and the binding reagents are antibodies specific forthe product of the enzymes with which they are co-immobilized.Alternatively, the binding reagents are broadly specific forphosphopeptides (e.g., an anti-phosphotyrosine or an anti-phosphoserineantibody). In another preferred embodiment, the enzymes are kinases, thesubstrates are the same and are a generic kinase substrate, and thebinding reagents are the same and are binding reagents broadly specificfor phosphopeptides (e.g., an anti-phosphotyrosine or ananti-phosphoserine antibody).

FIGS. 8A-8C illustrate expanded dynamic range binding assays, accordingto preferred embodiments of the invention, comprising three assaydomains of varying affinity for the analyte of interest. This assayformat is particularly advantageous when the dynamic range must extendto analyte concentrations that are greater than the binding capacity ofassay domains in a well. Well 800 in a MDMW Plate comprises assaydomains 810A-C for measuring analyte 820, the well comprising i) assaydomain 810A comprising immobilized binding reagent 815A, binding reagent815A having a dissociation constant for analyte=K_(d) ^(a), ii) assaydomain 810B comprising immobilized binding reagent 815B, binding reagent815B having a dissociation constant for analyte=K_(d) ^(b) and iii)assay domain 810C comprising immobilized binding reagent 815C, bindingreagent 815C having a dissociation constant for analyte=K_(d) ^(c),wherein K_(d) ^(a)<K_(d) ^(b)<K_(d) ^(c) and wherein K_(d) ^(b) andK_(d) ^(c) are, preferably, greater than the concentration of analyteneeded to saturate assay domain 810A. A labeled analyte 820 isintroduced into the well. Preferably, the dissociation constants differby a factor of 10 or more so that: i) when the concentration of analyteis <<K_(d) ^(b), only assay domain 810A will be significantly populated(FIG. 8A); ii) when the concentration of analyte is ˜K_(d) ^(b), assaydomain 810A will be saturated, assay domain 810B will be partiallypopulated, and assay domain 810C will be negligibly populated (FIG. 8B);and iii) when the concentration of analyte is ˜K_(d) ^(c), assay domains810A and 810B will be saturated and assay domain 810C will be partiallypopulated (FIG. 8C). In each concentration range, the signal from thepartially populated assay domain is used to quantitate analyte.Optionally, assay domain 810C is omitted or additional assay domains areincluded for extending the dynamic range into additional concentrationranges.

FIGS. 9A-9B illustrate expanded dynamic range binding assays, accordingto preferred embodiment of the inventions, comprising a sandwich bindingassay and a competitive binding assay for the same analyte. This assayformat is particularly advantageous when the dynamic range must extendto analyte concentrations that are greater than the binding capacity ofassay domains in a well. Well 900 in a MDMW Plate comprises i) assaydomain 910 comprising an immobilized capture binding reagent 912 that isspecific for analyte 915 and ii) assay domain 920 comprising animmobilized competitor 925 that competes with analyte 915 for binding tolabeled binding reagent 917. Introduction of analyte 915 and labeledbinding reagent 917 leads to the binding of labeled binding reagent 917to assay domain 910 via a sandwich complex and to assay domain 920 viadirect binding. Preferably, the amount of capture binding reagent 912 isroughly the same as the amount of competitor 925 and is roughly half ofthe amount of labeled binding reagent 917. For amounts of analyte lessthan the amount of capture binding reagent (i.e., the binding capacityof assay domain 910), the amount of label bound to assay domain 910 isroughly proportional to the amount of analyte and the amount of labelbound to assay domain 920 is roughly constant and saturated (FIG. 9A).For amounts of analyte greater than the amount of capture bindingreagent, the amount of label bound to assay domain 910 decrease withincreasing analyte due to the “hook effect” and the amount of labelbound to assay domain 920 also decreases due to competitive binding(FIG. 9B). The competitive assay can therefore be used to quantitateanalyte at high concentrations of analyte or simply to provide warningthat the sandwich assay has exceeded its dynamic range. In a preferredembodiment, analyte 915 is a protein and binding reagents 912 and 917are antibodies specific for different epitopes on analyte 915.Competitor 925 may be a labeled version of analyte 915 or, morepreferably, is a peptide that binds to binding reagent 917 but notbinding reagent 912 (thereby, reducing the possibility of bindingreagent 912 or competitor 925 leaching from the surface and binding).

According to preferred embodiments of the invention, the assay domainsof the invention are incorporated in assay modules or plates adapted forelectrode induced luminescence (preferably, electrochemiluminescence)assays, e.g., assay domains are supported on one or more integratedelectrodes within an assay cell (e.g., the well of a MDMW plate).Suitable assay modules and well plates, and methods of using such assaymodules and plates and systems incorporating the same are set forth inU.S. application Ser. Nos. 10/185,274 and 10/185,363, entitled “AssayPlates, Reader Systems and Methods for Luminescence Test Measurements”,filed Jun. 28, 2002 (see Sections 3, 4 and 5.1-5.6), hereby incorporatedby reference. According to one preferred embodiment of the invention, anassay module or plate comprises one or more (preferably two or more, 6or more, 24 or more, 96 or more, 384 or more, 1536 or more or 9600 ormore) assay wells, assay chambers and/or assay domains (e.g., discretelocations on a module surface where an assay reaction occurs and/orwhere an assay signal is emitted; typically an electrode surface,preferably a working electrode surface). According to an even morepreferred embodiment, the assay module is a multi-well assay platehaving a standard well configuration (e.g., 6 well, 24 well, 96 well,384 well, 1536 well, 6144 well or 9600 well). The wells of such platescan further comprise a plurality (e.g., 2 or more, 4 or more, 7 or more,25 or more, 64 or more, 100 or more, etc.) of discrete assay domains.

One aspect of the invention relates to improved assay modules (e.g.,plates) adapted for use in assays, preferably luminescence assays, morepreferably electrode induced luminescence assays, even more preferablyelectrochemiluminescence assays. The assay modules of the invention arepreferably suitable not only for ECL assays, but also suitable forfluorescence assays, chemiluminescence assays, bioluminescence assays,phosphorescence assays, optical transmittance assays (e.g., measurementsof optical density or light scattering) and electrochemical assays(e.g., wherein the measurement involves measuring current or voltage).

According to one preferred embodiment of the invention, an assay moduleor plate comprises one or more (preferably two or more, 6 or more, 24 ormore, 96 or more, 384 or more, 1536 or more or 9600 or more) assaywells, assay chambers and/or assay domains (e.g., discrete locations ona module surface where an assay reaction occurs and/or where an assaysignal is emitted; typically an electrode surface, preferably a workingelectrode surface). According to a particularly preferred embodiment,the assay plate is a multi-well assay plate having a standard wellconfiguration (e.g., 6 well, 24 well, 96 well, 384 well, 1536 well, 6144well or 9600 well).

An electrode induced luminescence well (preferablyelectrochemiluminescence well (i.e., a well adapted forelectrochemiluminescence)) or electrode induced luminescence domain(preferably electrochemiluminescence assay domain (i.e., an assay domainadapted for electrochemiluminescence assays)) may include a firstelectrode surface (such as a working electrode surface) and, preferablyalso includes a second electrode surface (such as a counter electrodesurface).

The invention also relates to a multi-well module, preferably an assayplate, for conducting one or more assays, the module having a pluralityof wells (and/or chambers), wherein two or more of the plurality ofwells (and/or chambers) comprise at least one first electrode surfaceand, preferably at least one counter electrode surface. According to apreferred embodiment, two or more of the plurality of wells (and/orchambers) comprise a working electrode surface and, preferably a counterelectrode surface, adapted to induce luminescence in the wells. Theinvention also relates to a multi-well module, preferably a plate, forconducting one or more assays, the module having a plurality of wells,wherein one or more of the plurality of wells comprise a workingelectrode surface and a counter electrode surface adapted to induceluminescence in the wells. Preferably, all or substantially all of thewells comprise an electrode surface.

Another embodiment relates to a multi-well assay module, preferably anassay plate, for conducting electrode induced luminescence (preferablyelectrochemiluminescence) assays, the module, preferably a plate, havinga plurality of wells, wherein each of the plurality of wells comprisesat least one first electrode surface (e.g., a working electrode) and,preferably, at least one second electrode surface (e.g., a counterelectrode).

Another embodiment relates to an assay plate for conducting one or moreelectrode induced luminescence (preferably electrochemiluminescence)assays, the plate having a plurality of wells or assay regionscomprising electrode surfaces, wherein the electrode surfaces consistessentially of at least one working electrode surface and at least onecounter electrode surface.

Preferably, the assay regions or assay wells are free of referenceelectrodes allowing for a greater density of assay domains andsimplified instrumentation for inducing and measuring luminescence.

One aspect of the invention relates to improved assay modules (e.g.,plates) adapted for use in assays, preferably luminescence assays, morepreferably electrode induced luminescence assays, even more preferablyelectrochemiluminescence assays. The assay modules of the invention arepreferably suitable not only for ECL assays, but also suitable forfluorescence assays, chemiluminescence assays, bioluminescence assays,phosphorescence assays, optical transmittance assays (e.g., measurementsof optical density or light scattering) and electrochemical assays(e.g., wherein the measurement involves measuring current or voltage).

According to one preferred embodiment of the invention, an assay moduleor plate comprises one or more (preferably two or more, 6 or more, 24 ormore, 96 or more, 384 or more, 1536 or more or 9600 or more) assaywells, assay chambers and/or assay domains (e.g., discrete locations ona module surface where an assay reaction occurs and/or where an assaysignal is emitted; typically an electrode surface, preferably a workingelectrode surface). According to a particularly preferred embodiment,the assay plate is a multi-well assay plate having a standard wellconfiguration (e.g., 6 well, 24 well, 96 well, 384 well, 1536 well, 6144well or 9600 well).

An electrode induced luminescence well (preferablyelectrochemiluminescence well (i.e., a well adapted forelectrochemiluminescence)) or electrode induced luminescence domain(preferably electrochemiluminescence assay domain (i.e., an assay domainadapted for electrochemiluminescence assays)) may include a firstelectrode surface (such as a working electrode surface) and, preferablyalso includes a second electrode surface (such as a counter electrodesurface).

The invention also relates to a multi-well module, preferably an assayplate, for conducting one or more assays, the module having a pluralityof wells (and/or chambers), wherein two or more of the plurality ofwells (and/or chambers) comprise at least one first electrode surfaceand, preferably at least one counter electrode surface. According to apreferred embodiment, two or more of the plurality of wells (and/orchambers) comprise a working electrode surface and, preferably a counterelectrode surface, adapted to induce luminescence in the wells. Theinvention also relates to a multi-well module, preferably a plate, forconducting one or more assays, the module having a plurality of wells,wherein one or more of the plurality of wells comprise a workingelectrode surface and a counter electrode surface adapted to induceluminescence in the wells. Preferably, all or substantially all of thewells comprise an electrode surface.

Another embodiment relates to a multi-well assay module, preferably anassay plate, for conducting electrode induced luminescence (preferablyelectrochemiluminescence) assays, the module, preferably a plate, havinga plurality of wells, wherein each of the plurality of wells comprisesat least one first electrode surface (e.g., a working electrode) and,preferably, at least one second electrode surface (e.g., a counterelectrode).

Another embodiment relates to an assay plate for conducting one or moreelectrode induced luminescence (preferably electrochemiluminescence)assays, the plate having a plurality of wells or assay regionscomprising electrode surfaces, wherein the electrode surfaces consistessentially of at least one working electrode surface and at least onecounter electrode surface.

Preferably, the assay regions or assay wells are free of referenceelectrodes allowing for a greater density of assay domains andsimplified instrumentation for inducing and measuring luminescence.

The working electrode surface area may be smaller, the same or largerthan the counter electrode surface area. In some embodiments, theworking electrode surface is preferably much larger than the counterelectrode surface. This configuration allows for a greater workingelectrode surface on which to immobilize assay reagents. Preferably, thesurface ratio of the working electrode surface to the counter electrodesurface is at least 2 to 1, more preferably at least 5 to 1, even morepreferably at least 10 to 1, still more preferred at least 50 to 1, evenmore preferably at least 100 to 1 and most preferred at least 500 to 1.Surprisingly, the assay modules of the invention provide for theperformance of electrochemiluminescence assays with very little counterelectrode surface. Preferably, the working electrode is substantiallycentered within the well so as to maximize the percentage of ECL emittedfrom the electrode that can be captured by a light detector placed abovethe well.

According to another embodiment, the first electrode surface (e.g.,working electrode surface) is centered at the bottom of each well andthe second electrode surface (e.g., counter electrode surface) isadjacent the periphery of the bottom of each well. In some embodiments,the working electrode surface is centered at the bottom of each well andis completely surrounded by the counter electrode surface.

Alternatively, for some applications it is desirable that workingelectrode surfaces be small, e.g., relative to the surface area of awell or well bottom. In some applications, this configuration may reducenon-specific signals. According to one embodiment of the invention, themulti-well assay module has a plurality of wells, each well having awell bottom comprising a first electrode surface, a second electrodesurface and a dielectric surface (preferably the dielectric surface isthe surface of the bottom of the well between the first electrodesurface and the second electrode surface), wherein the ratio of thefirst electrode surface and the dielectric surface (or alternatively thesurface of the well bottom) is less than 1 to 5, preferably 1 to 10,more preferably 1 to 30.

According to one preferred embodiment of the invention, the assay modulecomprises a first electrode surface (preferably a working electrodesurface) that is bounded by a dielectric surface, the dielectric surfacebeing raised or lowered (preferably, raised) and/or of differenthydrophobicity (preferably, more hydrophobic) than the electrodesurface. Preferably, the dielectric boundary is higher, relative to theelectrode surface, by 0.5-100 micrometers, or more preferably by 2-30micrometers, or most preferably by 8-12 micrometers. Even morepreferably, the dielectric boundary has a sharply defined edge (i.e.,providing a steep boundary wall and/or a sharp angle at the interfacebetween the electrode and the dielectric boundary). Preferably, thefirst electrode surface has a contact angle for water 10 degrees lessthan the dielectric surface, preferably 15 degrees less, more preferably20 degrees less, more preferably 30 degrees less, even more preferably40 degrees less, and most preferred 50 degrees less. One advantage ofhaving a dielectric surface that is raised and/or more hydrophobic thanthe electrode surface is in the reagent deposition process where thedielectric boundary may be used to confine a reagent within the boundaryof the electrode surface. In particular, having a sharply defined edgewith a steep boundary wall and/or a sharp angle at the interface betweenthe electrode and dielectric boundary is especially useful for “pinning”drops of solution and confining them to the electrode surface.

According to another embodiment, an assay module comprises one or more(preferably two or more) wells, the wells having one or more firstelectrode surfaces (preferably one or more working electrode surfaces)and a plurality of assay domains immobilized therein. Preferably, atleast two of the plurality of the assay domains comprises differentbinding reagents. Preferably, each well comprises at least four, morepreferably at least seven, even more preferably at least ten assaydomains and most preferred at least 15 assay domains. One preferredembodiment is a 24 well plate wherein each well comprises at least 16,preferably at least 25, more preferably at least 64, even morepreferably at least 100 assay domains per well and most preferably atleast 250 assay domains per well.

Another embodiment of the invention relates to a multi-well module(preferably a multi-well plate) having a plurality of wells, wherein thewells comprise a plurality of working electrode surfaces having assaydomains immobilized thereon. Preferably, the assay domains areindependently addressable. For example, a well may comprise a pluralityof assay domains, wherein each assay domain comprises an electrode whichis independently addressable from the other assay domains within thewell. In another example, a group of wells may each comprise a pluralityof assay domains, wherein each assay domain comprises an electrode whichis independently addressable from the other assay domains within thewell, but which is jointly addressable with an assay domain in each ofthe other wells.

Alternatively, for some applications it is desirable that workingelectrode surfaces be small, e.g., relative to the surface area of awell or well bottom. In some applications, this configuration may reducenon-specific signals. According to one embodiment of the invention, themulti-well assay module has a plurality of wells, each well having awell bottom comprising a first electrode surface, a second electrodesurface and a dielectric surface (preferably the dielectric surface isthe surface of the bottom of the well between the first electrodesurface and the second electrode surface), wherein the ratio of thefirst electrode surface and the dielectric surface (or alternatively thesurface of the well bottom) is less than 1 to 5, preferably 1 to 10,more preferably 1 to 30.

According to one preferred embodiment of the invention, the assay modulecomprises a first electrode surface (preferably a working electrodesurface) that is bounded by a dielectric surface, the dielectric surfacebeing raised or lowered (preferably, raised) and/or of differenthydrophobicity (preferably, more hydrophobic) than the electrodesurface. Preferably, the dielectric boundary is higher, relative to theelectrode surface, by 0.5-100 micrometers, or more preferably by 2-30micrometers, or most preferably by 8-12 micrometers. Even morepreferably, the dielectric boundary has a sharply defined edge (i.e.,providing a steep boundary wall and/or a sharp angle at the interfacebetween the electrode and the dielectric boundary). Preferably, thefirst electrode surface has a contact angle for water 10 degrees lessthan the dielectric surface, preferably 15 degrees less, more preferably20 degrees less, more preferably 30 degrees less, even more preferably40 degrees less, and most preferred 50 degrees less. One advantage ofhaving a dielectric surface that is raised and/or more hydrophobic thanthe electrode surface is in the reagent deposition process where thedielectric boundary may be used to confine a reagent within the boundaryof the electrode surface. In particular, having a sharply defined edgewith a steep boundary wall and/or a sharp angle at the interface betweenthe electrode and dielectric boundary is especially useful for “pinning”drops of solution and confining them to the electrode surface.

According to another embodiment, an assay module comprises one or more(preferably two or more) wells, the wells having one or more firstelectrode surfaces (preferably one or more working electrode surfaces)and a plurality of assay domains immobilized therein. Preferably, atleast two of the plurality of the assay domains comprises differentbinding reagents. Preferably, each well comprises at least four, morepreferably at least seven, even more preferably at least ten assaydomains and most preferred at least 15 assay domains. One preferredembodiment is a 24 well plate wherein each well comprises at least 16,preferably at least 25, more preferably at least 64, even morepreferably at least 100 assay domains per well and most preferably atleast 250 assay domains per well.

Another embodiment of the invention relates to a multi-well module(preferably a multi-well plate) having a plurality of wells, wherein thewells comprise a plurality of working electrode surfaces having assaydomains immobilized thereon. Preferably, the assay domains areindependently addressable. For example, a well may comprise a pluralityof assay domains, wherein each assay domain comprises an electrode whichis independently addressable from the other assay domains within thewell. In another example, a group of wells may each comprise a pluralityof assay domains, wherein each assay domain comprises an electrode whichis independently addressable from the other assay domains within thewell, but which is jointly addressable with an assay domain in each ofthe other wells.

The invention also relates to methods and apparatus for the measurementof signals from assay modules and MDMW plates of the invention. Thepreferred apparatus of the invention can be used to induce and measureluminescence in assays conducted in assay modules, preferably inmulti-well assay plates. It may incorporate, for example, one or morephotodetectors; a light tight enclosure; electrical connectors forcontacting the assay modules; mechanisms to transport multi-well assaymodules into and out of the apparatus (and in particular, into and outof light tight enclosures); mechanisms to align and orient multi-wellassay modules with the photodetector(s) and with electrical contacts;mechanisms to track and identify modules (e.g. bar code readers);mechanisms to make electrical connections to modules, one or moresources of electrical energy for inducing luminescence in the modules;and appropriate electronics and software.

The apparatus may also include mechanisms to store, stack, move and/ordistribute one or more assay modules (e.g. multi-well plate stackers).The apparatus may advantageously use arrays of photodetectors (e.g.arrays of photodiodes) or imaging photodetectors (e.g. CCD cameras) tomeasure light. These detectors allow the apparatus to measure the lightfrom multiple wells, assay domains, and/or assay cells simultaneouslyand/or to image the intensity and spatial distribution of light emittedfrom an individual well, assay cell and/or assay domain.

The apparatus can preferably measure light from one or more sectors ofan assay module, preferably a multi-well assay plate. In someembodiments, a sector comprises a group of wells, assay domains and/orassay cells numbering between one and a number fewer than the totalnumber of wells (and/or chambers) in the assay module (e.g. a row,column, or two-dimensional sub-array of wells in a multi-well plate). Inpreferred embodiments, a sector comprises between 4 percent and 50percent of the wells of a multi-well plate. In especially preferredembodiments, multi-well assay plates are divided into columnar sectors(each sector having one row or column of wells) or square sectors (e.g.,a standard sized multi-well plate can be divided into six square sectorsof equal size). In some embodiments, a sector may comprise one or morewells with more than one fluid containment region within the wells. Theapparatus, preferably, is adapted to sequentially induce ECL in and/orsequentially measure ECL from the sectors in a given module, preferablyplate.

One aspect of the invention relates to the immobilization of materialsin assay domains on electrodes having improved electrode compositionsand surfaces and assay modules comprising these electrode compositionsand surfaces. Electrodes in the present invention are preferablycomprised of a conductive material. The electrode may comprise a metalsuch as gold, silver, platinum, nickel, steel, iridium, copper,aluminum, a conductive alloy, or the like. They may also comprise oxidecoated metals (e.g. aluminum oxide coated aluminum) Electrodes maycomprise non-metallic conductors such as conductive forms of molecularcarbon. Electrodes may also be comprised of semiconducting materials(e.g. silicon, germanium) or semi-conducting films such as indium tinoxide (ITO), antimony tin oxide (ATO) and the like. Electrodes may alsobe comprised of mixtures of materials containing conducting composites,inks, pastes, polymer blends, metal/non-metal composites and the like.Such mixtures may include conductive or semi-conductive materials mixedwith non-conductive materials. Preferably, electrode materials aresubstantially free of silicone-based materials.

Electrodes (in particular working electrodes) used in assay modules ofthe invention are advantageously able to induce luminescence fromluminescent species. Preferable materials for working electrodes arematerials able to induce electrochemiluminescence fromRuthenium-tris-bipyridine in the presence of tertiary alkyl amines (suchas tripropyl amine) Examples of such preferred materials includeplatinum, gold, ITO, carbon, carbon-polymer composites, and conductivepolymers.

Preferably, electrodes are comprised of carbon-based materials such ascarbon, carbon black, graphitic carbon, carbon nanotubes, carbonfibrils, graphite, carbon fibers and mixtures thereof. Advantageously,they may be comprised of conducting carbon-polymer composites,conducting particles dispersed in a matrix (e.g. carbon inks, carbonpastes, metal inks), and/or conducting polymers. One preferredembodiment of the invention is an assay module, preferably a multi-wellplate, having electrodes (e.g., working and/or counter electrodes) thatcomprise carbon, preferably carbon layers, more preferablyscreen-printed layers of carbon inks Some useful carbon inks includematerials produced by Acheson Colloids Co. (e.g., Acheson 440B, 423ss,PF407A, PF407C, PM-003A, 30D071, 435A, Electrodag 505SS, and Aquadag™),E. I. Du Pont de Nemours and Co. (e.g., Dupont 7105, 7101, 7102, 7103,7144, 7082, 7861D, and CB050), Conductive Compounds Inc (e.g., C-100),and Ercon Inc. (e.g., G-451).

In another preferred embodiment, the electrodes of the inventioncomprise carbon fibrils. The terms “carbon fibrils”, “carbon nanotubes”,single wall nanotubes (SWNT), multiwall nanotubes (MWNT), “graphiticnanotubes”, “graphitic fibrils”, “carbon tubules”, “fibrils” and“buckeytubes”, all of which terms may be used to describe a broad classof carbon materials (see Dresselhaus, M. S.; Dresselhaus, G.; Eklund, P.C.; “Science of Fullerenes and Carbon Nanotubes”, Academic Press, SanDiego, Calif., 1996, and references cited therein). The terms “fibrils”and “carbon fibrils” are used throughout this application to includethis broad class of carbon-based materials. Individual carbon fibrils asdisclosed in U.S. Pat. Nos. 4,663,230; 5,165,909; and 5,171,560 areparticularly advantageous. They may have diameters that range from about3.5 nm to 70 nm, and length greater than 10² times the diameter, anouter region of multiple, essentially continuous, layers of orderedcarbon atoms and a distinct inner core region. Simply for illustrativepurposes, a typical diameter for a carbon fibril may be approximatelybetween about 7 and 25 nm, and a typical range of lengths may be 1000 nmto 10,000 nm. Carbon fibrils may also have a single layer of carbonatoms and diameters in the range of 1 nm-2 nm. Electrodes of theinvention may comprise one or more carbon fibrils, e.g., in the form ofa fibril mat, a fibril aggregate, a fibril ink, a fibril composite(e.g., a conductive composite comprising fibrils dispersed in an oil,paste, ceramic, polymer, etc.). One preferred embodiment of theinvention relates to a multi-well plate comprising a substratecomprising a carbon nanotube-containing composite (preferably, carbonnanotubes dispersed in a polymeric matrix), wherein the surface of thesubstrate is etched to expose the carbon nanotubes, thereby forming oneor more working electrodes.

Electrodes may be formed into patterns by a molding process (i.e.,during fabrication of the electrodes), by patterned deposition, bypatterned printing, by selective etching, through a cutting process suchas die cutting or laser drilling, and/or by techniques known in the artof electronics microfabrication. Electrodes may be self supporting ormay be supported on another material, e.g. on films, plastic sheets,adhesive films, paper, backings, meshes, felts, fibrous materials, gels,solids (e.g. metals, ceramics, glasses), elastomers, liquids, tapes,adhesives, other electrodes, dielectric materials and the like. Thesupport may be rigid or flexible, flat or deformed, transparent,translucent, opaque or reflective. Preferably, the support comprises aflat sheet of plastic such as acetate or polystyrene. Electrodematerials may be applied to a support by a variety of coating anddeposition processes known in the art such as painting, spray-coating,screen-printing, ink-jet printing, laser printing, spin-coating,evaporative coating, chemical vapor deposition, etc. Supportedelectrodes may be patterned using photolithographic techniques (e.g.,established techniques in the microfabrication of electronics), byselective etching, and/or by selective deposition (e.g., by evaporativeor CVD processes carried out through a mask). In a preferred embodiment,electrodes are comprised of extruded films of conducting carbon/polymercomposites. In another preferred embodiment, electrodes are comprised ofa screen printed conducting ink deposited on a substrate. Electrodes maybe supported by another conducting material. Advantageously, screenprinted carbon ink electrodes are printed over a conducting metal ink(e.g., silver ink) layer so as to improve the conductivity of theelectrodes.

According to one preferred embodiment of the invention, the electrodesurface (preferably a working electrode surface of an assay module orassay plate) is bounded by a dielectric surface, the dielectric surfacebeing raised or lowered (preferably, raised) and/or of differenthydrophobicity (preferably, more hydrophobic) than the electrodesurface. Preferably, the dielectric boundary is higher, relative to theelectrode surface, by 0.5-100 micrometers, or more preferably by 2-30micrometers, or most preferably by 8-12 micrometers. Even morepreferably, the dielectric boundary has a sharply defined edge (i.e.,providing a steep boundary wall and/or a sharp angle at the interfacebetween the electrode and the dielectric boundary).

Preferably, the first electrode surface has a contact angle for water 10degrees less than the dielectric surface, preferably 15 degrees less,more preferably 20 degrees less, more preferably 30 degrees less, evenmore preferably 40 degrees less, and most preferred 50 degrees less. Oneadvantage of having a dielectric surface that is raised and/or morehydrophobic than the electrode surface is in the reagent depositionprocess where the dielectric boundary may be used to confine a reagentwithin the boundary of the electrode surface. In particular, having asharply defined edge with a steep boundary wall and/or a sharp angle atthe interface between the electrode and dielectric boundary isespecially useful for “pinning” drops of solution and confining them tothe electrode surface. In an especially preferred embodiment of theinvention, the dielectric boundary is formed by printing a patterneddielectric ink on and/or around the electrode, the pattern designed soas to expose one or more assay domains on the electrode.

Electrodes may be modified by chemical or mechanical treatment toimprove the immobilization of reagents. The surface may be treated tointroduce functional groups for immobilization of reagents or to enhanceits adsorptive properties. Surface treatment may also be used toinfluence properties of the electrode surface, e.g., the spreading ofwater on the surface or the kinetics of electrochemical processes at thesurface of the electrode. Techniques that may be used include exposureto electromagnetic radiation, ionizing radiation, plasmas or chemicalreagents such as oxidizing agents, electrophiles, nucleophiles, reducingagents, strong acids, strong bases and/or combinations thereof.Treatments that etch one or more components of the electrodes may beparticularly beneficial by increasing the roughness and therefore thesurface area of the electrodes. In the case of composite electrodeshaving conductive particles or fibers (e.g., carbon particles orfibrils) in a polymeric matrix or binder, selective etching of thepolymer may be used to expose the conductive particles or fibers.

One particularly useful embodiment is the modification of the electrode,and more broadly a material incorporated into the present invention bytreatment with a plasma, specifically a low temperature plasma, alsotermed glow-discharge. The treatment is carried out in order to alterthe surface characteristics of the electrode, which come in contact withthe plasma during treatment. Plasma treatment may change, for example,the physical properties, chemical composition, or surface-chemicalproperties of the electrode. These changes may, for example, aid in theimmobilization of reagents, reduce contaminants, improve adhesion toother materials, alter the wettability of the surface, facilitatedeposition of materials, create patterns, and/or improve uniformity.Examples of useful plasmas include oxygen, nitrogen, argon, ammonia,hydrogen, fluorocarbons, water and combinations thereof. Oxygen plasmasare especially preferred for exposing carbon particles in carbon-polymercomposite materials. Oxygen plasmas may also be used to introducecarboxylic acids or other oxidized carbon functionality into carbon ororganic materials (these may be activated, e.g., as active esters oracyl chlorides) so as to allow for the coupling of reagents. Similarly,ammonia-containing plasmas may be used to introduce amino groups for usein coupling to assay reagents.

Treatment of electrode surfaces may be advantageous so as to improve orfacilitate immobilization, change the wetting properties of theelectrode, increase surface area, increase the binding capacity for theimmobilization of reagents (e.g., lipid, protein or lipid/proteinlayers) or the binding of analytes, and/or alter the kinetics ofelectrochemical reactions at the electrode. In some applications,however, it may be preferable to use untreated electrodes. For example,we have found that it is advantageous to etch carbon ink electrodesprior to immobilization when the application calls for a large dynamicrange and therefore a high binding capacity per area of electrode. Wehave discovered that oxidative etching (e.g., by oxygen plasma) hasadditional advantages in that the potential for oxidation of tripropylamine (TPA) and the contact angle for water are both reduced relative tothe unetched ink. The low contact angle for water allows reagents to beadsorbed on the electrode by application of the reagents in a smallvolume of aqueous buffer and allowing the small volume to spread evenlyover the electrode surface. Surprisingly, we have found that excellentassays may also be carried out on unetched carbon ink electrodes despitethe presence of polymeric binders in the ink. In fact, in someapplications requiring high sensitivity or low-non specific binding itis preferred to use unetched carbon ink electrodes so as to minimize thesurface area of exposed carbon and therefore minimize background signalsand loss of reagents from non-specific binding of reagents to theexposed carbon. Depending on the ink used and the process used to applythe ink, the electrode surface may not be easily wettable by aqueoussolutions. We have found that we can compensate for the low wettabilityof the electrodes during the adsorption of reagents by adding lowconcentrations of non-ionic detergents to the reagent solutions so as tofacilitate the spreading of the solutions over the electrode surface.Even spreading is especially important during the localizedimmobilization of a reagent from a small volume of solution. Forexample, we have found that the addition of 0.005-0.04% Triton® X-100allows for the spreading of protein solutions over unetched carbon inksurfaces without affecting the adsorption of the protein to theelectrode and without disrupting the ability of a dielectric filmapplied on or adjacent to the electrode (preferably, a printeddielectric film with a thickness of 0.5-100 micrometers, or morepreferably 2-30 micrometers, or most preferably 8-12 micrometers andhaving a sharply defined edge) to confine fluids to the electrodesurface. Preferably, when non-ionic detergents such as Triton® X-100 areused to facilitate spreading of reagents (e.g., capture reagents) ontounetched screen-printed electrodes (i.e., so as to allow theimmobilization of the reagents), the solutions containing the reagentsare allowed to dry onto the electrode surface. It has been found thatthis drying step greatly improves the efficiency and reproducibility ofthe immobilization process.

Electrodes can be derivatized with chemical functional groups that canbe used to attach other materials to them. Materials may be attachedcovalently to these functional groups, or they may be adsorbednon-covalently to derivatized or underivatized electrodes. Electrodesmay be prepared with chemical functional groups attached covalently totheir surface. These chemical functional groups include but are notlimited to COOH, OH, NH₂, activated carboxyls (e.g., N-hydroxysuccinimide (NHS)-esters), poly-(ethylene glycols), thiols, alkyl((CH₂)_(n)) groups, and/or combinations thereof). Certain chemicalfunctional groups (e.g., COOH, OH, NH₂, SH, activated carboxyls) may beused to couple reagents to electrodes. For further reference to usefulimmobilization and bioconjugation techniques see G. Hermanson, A. Malliaand P. Smith, Immobilized Affinity Ligand Techniques (Academic Press,San Diego, 1992) and G. Hermanson, Bioconjugate Techniques (AcademicPress, San Diego, 1996).

In preferred embodiments, NHS-ester groups are used to attach othermolecules or materials bearing a nucleophilic chemical functional group(e.g., an amine). In a preferred embodiment, the nucleophilic chemicalfunctional group is present on and/or in a biomolecule, either naturallyand/or by chemical derivatization. Examples of suitable biomoleculesinclude, but are not limited to, amino acids, proteins and functionalfragments thereof, antibodies, binding fragments of antibodies, enzymes,nucleic acids, and combinations thereof. This is one of many suchpossible techniques and is generally applicable to the examples givenhere and many other analogous materials and/or biomolecules. In apreferred embodiment, reagents that may be used for ECL may be attachedto the electrode via NHS-ester groups.

It may be desirable to control the extent of non-specific binding ofmaterials to electrodes. Simply by way of non-limiting examples, it maybe desirable to reduce or prevent the non-specific adsorption ofproteins, antibodies, fragments of antibodies, cells, subcellularparticles, viruses, serum and/or one or more of its components, ECLlabels (e.g., Ru^(II)(bpy)₃ and Ru^(III)(bpy)₃ derivatives), oxalates,trialkylamines, antigens, analytes, and/or combinations thereof). Inanother example, it may be desirable to enhance the binding ofbiomolecules.

One or more chemical moieties that reduce or prevent non-specificbinding (also known as blocking groups) may be present in, on, or inproximity to an electrode. Such moieties, e.g., PEG moieties and/orcharged residues (e.g., phosphates, ammonium ions), may be attached toor coated on the electrode. Examples of useful blocking reagents includeproteins (e.g., serum albumins and immunoglobins), nucleic acids,polyethylene oxides, polypropylene oxides, block copolymers ofpolyethylene oxide and polypropylene oxide, polyethylene imines anddetergents or surfactants (e.g., classes of non-ionicdetergents/surfactants known by the trade names of Brij, Triton, Tween,Thesit, Lubrol, Genapol, Pluronic (e.g., F108), Tetronic, Tergitol, andSpan).

Materials used in electrodes may be treated with surfactants to reducenon-specific binding. For example, electrodes may be treated withsurfactants and/or detergents that are well known to one of ordinaryskill in the art (for example, the Tween, Triton, Pluronics (e.g.,F108), Span, and Brij series of detergents). Solutions of PEGs and/ormolecules which behave in similar fashion to PEG (e.g., oligo- orpolysaccharides, other hydrophilic oligomers or polymers) (“Polyethyleneglycol chemistry: Biotechnical and Biomedical Applications”, Harris, J.M. Editor, 1992, Plenum Press) may be used instead of and/or inconjunction with surfactants and/or detergents. Undesirable non-specificadsorption of certain entities such as those listed above may be blockedby competitive non-specific adsorption of a blocking agent, e.g., by aprotein such as bovine serum albumin (BSA) or immunoglobulin G (IgG).One may adsorb or covalently attach an assay reagent on an electrode andsubsequently treat the electrode with a blocking agent so as to blockremaining unoccupied sites on the surface.

In preferred embodiments, it may be desirable to immobilize (by eithercovalent or non-covalent means) biomolecules or other media tocarbon-containing materials, e.g., carbon black, fibrils, and/or carbondispersed in another material. One may attach antibodies, fragments ofantibodies, proteins, enzymes, enzyme substrates, inhibitors, cofactors,antigens, haptens, lipoproteins, liposaccharides, cells, sub-cellularcomponents, cell receptors, viruses, nucleic acids, antigens, lipids,glycoproteins, carbohydrates, peptides, amino acids, hormones,protein-binding ligands, pharmacological agents, and/or combinationsthereof. It may also be desirable to attach non-biological entities suchas, but not limited to polymers, elastomers, gels, coatings, ECL tags,redox active species (e.g., tripropylamine, oxalates), inorganicmaterials, chelating agents, linkers, etc. A plurality of species may beco-adsorbed to form a mixed layer on the surface of an electrode. Mostpreferably, biological materials (e.g., proteins) are immobilized oncarbon-containing electrodes by passive adsorption. Surprisingly,biological membranes (e.g., cells, cell membranes, membrane fragments,membrane vesicles, lipsomes, organelles, viruses, bacteria, etc.) may bedirectly adsorbed on carbon without destroying the activity of membranecomponents or their accessibility to binding reagents (see, e.g.,copending U.S. application Ser. No. 10/208,526 (entitled “AssayElectrodes Having Immobilized Lipid/Protein Layers, Methods Of MakingThe Same And Methods Of Using The Same For Luminescence TestMeasurements”), filed on Jul. 29, 2002, hereby incorporated byreference.

Electrodes used in the multi-well assay plates of the invention aretypically non-porous, however, in some applications it is advantageousto use porous electrodes (e.g., mats of carbon fibers or fibrils,sintered metals, and metals films deposited on filtration membranes,papers or other porous substrates. These applications include those thatemploy filtration of solutions through the electrode so as to: i)increase mass transport to the electrode surface (e.g., to increase thekinetics of binding of molecules in solution to molecules on theelectrode surface); ii) capture particles on the electrode surface;and/or iii) remove liquid from the well.

The assay modules of the present invention may use dielectric inks,films or other electrically insulating materials (hereinafter referredto as dielectrics). Dielectrics in the present invention may be used toprevent electrical connectivity between electrodes, to define patternedregions, to adhere materials together (i.e., as adhesives), to supportmaterials, to define assay domains, as masks, as indicia and/or tocontain assay reagents and other fluids. Dielectrics are non-conductingand advantageously non-porous (i.e., do not permit transmission ofmaterials) and resistant to dissolving or degrading in the presence ofmedia encountered in an electrode induced luminescence measurement. Thedielectrics in the present invention may be liquids, gels, solids ormaterials dispersed in a matrix. They may be deposited in uncured formand cured to become solid. They may be inks, solid films, tapes orsheets. Materials used for dielectrics include polymers, photoresists,plastics, adhesives, gels, glasses, non-conducting inks, non-conductingpastes, ceramics, papers, elastomers, silicones, thermoplastics.Preferably, dielectric materials of the invention are substantially freeof silicones. Examples of non-conducting inks include UV curabledielectrics such as materials produced by Acheson Colloids Co. (e.g.,Acheson 451SS, 452SS, PF-021, ML25251, ML25240, ML25265, and Electrodag38DJB16 clear) and E. I. du Pont de Nemours and Co. (e.g., Dupont: 5018,3571, and 5017).

Dielectrics of the present invention may be applied by a variety ofmeans, for example, printing, spraying, laminating, or may be affixedwith adhesives, glues, solvents or by use of mechanical fasteners.Patterns and/or holes in dielectric layers may be formed by moldingprocesses (i.e., during fabrication of the layer), by selective etchingand/or by a cutting process such as die cutting or laser drilling.Dielectrics may be deposited and/or etched in patterns through the useof established photolithographic techniques (e.g., techniques used inthe semiconductor electronics industry) and/or by patterned depositionusing an evaporative or CVD process (e.g., by deposition through amask). In a preferred embodiment, a dielectric ink is deposited on asubstrate by printing (e.g., ink jet printing, laser printing or, morepreferably, screen printing) and, optionally, UV cured. Preferably, thescreen printed dielectric is UV curable allowing for improved edgedefinition than solvent based dielectrics. In another preferredembodiment, a non-conducting polymeric film is affixed to a supportusing an adhesive.

When using a dielectric ink printed on or adjacent an electrode toconfine fluids to regions of the electrode surface, the dielectric filmpreferably has a thickness of 0.5-100 micrometers, or more preferably2-30 micrometers, or most preferably 8-12 micrometers and also,preferably, has a sharply defined edge with steep walls.

The invention includes plate tops and assembled plates comprising aplate top and, preferably, a plate bottom defining well bottoms havingone or more electrode surfaces, most preferably having one or moreworking electrode surfaces and, optionally, one or more counterelectrode surfaces. Preferably, the plate top is a structure with holes,wherein the structure may be combined with a plate bottom to form amulti-well plate, the walls of the wells of the plate being at leastpartially defined by the inside surfaces of the holes through the platetop. The holes through the plate top may be a variety of shapes (e.g.,round, oval, square, rectangular, triangular, star shaped, etc.). Theholes may be of various sizes. They can also have irregular dimensionswithin a hole (e.g., the hole may become more narrow or more wide atdifferent depths). For example, the hole may be shaped like a cone,becoming more narrow at the bottom so as to optimize the collection oflight emitted from the well bottom. The plate top may also havestructures or indicia thereon that aid in identifying the plate top,distinguishing the plate top from other configurations of plate top, orin aligning and handling the plate top. Advantageously, the dimensionsand structure of the plate top are preferably in accordance with, or atleast compatible with, industry standards for the footprints and shapesof assay plates.

The plate top may be made from conducting or non-conducting materials.Preferably, the majority of the plate top is a unitary molded structuremade from rigid thermoplastic material such as polystyrene, polyethyleneor polypropylene. Optimally, this unitary structure is formed of (or,alternatively, coated with) inexpensive material that is generallyimpervious to reactants, can withstand modest levels of heat and lightand is, preferably, resistant to the adsorption of biomolecules.Preferably, the plate top is substantially free of silicones. Plate topsmay be clear or translucent. Different colored materials may be used toimprove the results of certain ECL measurement processes.

It is preferable that the plate top comprise a material that does nottransmit light so as to prevent cross-talk between wells. A highlyreflective metallic coating or constituent material may provide anespecially reflective interior surface for each of wells so as toincrease the efficiency with which light can be transmitted tophotodetectors. An opaque white plastic material such as a plasticfilled with light scattering particles (e.g., lead oxide, alumina,silica or, preferably, titanium dioxide particles) may provide aninterior surface for the wells that is highly light scattering therebyimproving light gathering efficiency. In one embodiment, preferred platetops comprise plastics (e.g., well walls) comprising such lightscattering particles at a concentration of from 4-20 wt %, preferably6-20%, more preferably 6-15%, even more preferably 6-12%, and mostpreferred approximately 9%. In an alternate preferred embodiment, theplate top comprises an opaque, preferably non-reflective, black materialto prevent the reflection or scattering of ECL-generated light fromdifferent locations within a well and to prevent reflective interferenceduring ECL test measurements. In general, when imaging light emittedfrom a well (e.g., when using a camera to produce an image of lightemitted from the well) it is advantageous that the interior surface ofthe well (e.g., as defined by a plate top) comprise an absorptive (e.g.,black) preferably non-scattering material since the detection ofscattered light will reduce the fidelity of the image. In general, whendetecting light in a non-imaging mode (e.g., when a single lightdetector is used to detect all the light emitted from a well) it isadvantageous that the interior surface of the well comprise a reflectiveor highly scattering material so as to prevent the loss of light due toadsorption of light at the well walls and to maximize the collection oflight at the detector.

The invention also includes assay module tops and assembled assaymodules comprising an assay module top and a plate bottom or assaymodule substrate. The assay module top may be a plate top (as describedabove). The assay module top may have, e.g., holes, channels, and/orwells that when mated to a plate bottom or assay module substrate definewells and/or chambers, such wells and/or chambers preferably comprisingone or more electrodes (and/or assay domains) provided by the platebottom or assay module substrate. The assay module top may haveadditional channels, tubes or other microfluidics so as to allow theflow of samples into, out of and/or between wells, flow cells andchambers of an assay module.

FIGS. 10A and 10B show a layered view and a stylized cross-sectionalview, respectively, of an embodiment of the multi-well assay plate ofthe invention. Multi-well assay plate 1000 is a laminar structurecomprising, in sequence, a plate top 1020, an adhesive layer 1030, adielectric layer 1040, a conductive layer 1050, a substrate layer 1060and a contact layer 1070. Holes 1022 and 1032 through plate top 1020 andadhesive layer 1030, respectively, are aligned so as to form a pluralityof wells 1002 having well bottoms defined by dielectric layer 1040,conductive layer 1050 and/or substrate layer 1060 and well walls definedby the interior surfaces of holes 1022 and 1032. Through-holes 1062 and1064 through substrate layer 1060 provide an electrical path betweenelements of conductive layer 1050 and elements of contact layer 1070.Details A-D show the pattern of layers 1070, 1060, 1050 and 1040 withina given sector of plate 1000. Element 1080 shows layers 1070, 1060, 1050and 1040 aligned and stacked, in order from top to bottom—1040 (top),1050, 1060, and 1070 (bottom)—so as to form a plate bottom withintegrated electrodes.

Plate top 1020 is a plate top as described above. Adhesive layer 1030 isan adhesive suitable for forming a fluid-tight seal between plate top1020 and dielectric layer 1040, conductive layer 1050 and/or substratelayer 1060. Adhesive layer 1030 may be an adhesive coating applied,e.g., by spray coating, onto plate top 1020. In a preferred embodiment,adhesive layer 1030 is a double sided adhesive tape (i.e., a plasticfilm coated on both sides with adhesive). Holes 1032 are preferablyformed by a cutting process such as laser drilling or die cutting.Optionally, adhesive 1030 may be omitted (e.g., when the adjoininglayers have adhesive properties or when sealing is accomplished withoutthe use of adhesives, e.g., by clamping, heat sealing, sonic welding,solvent welding, etc.). Alternatively, both plate top 1020 and adhesivelayer 1030 may be omitted.

Conductive layer 1050 comprises materials suitable for use as workingelectrodes and/or counter electrodes in an ECL assay and is supported onsubstrate 1060, a non-conductive substrate such as a plastic sheet orfilm. Preferably, conductive layer 1050 is a conductive coating such asa carbon ink and may be formed by a printing process such as screenprinting. Conductive layer 1050 is sectioned, e.g., by screen printingin a defined pattern, into 6 electrically isolated working electrodesections 1052 and 6 electrically isolated counter electrode sections1054 so as to divide plate 1000 into 6 independently addressable squaresectors. As shown in the figure, the sectioning is designed so thatfluid in a given well will be in contact with at least one workingelectrode section and at least one counter electrode section. Theworking electrode sections may have a different composition than thecounter electrode sections so as to optimize the performance of theelectrodes or they may comprise the same materials so as to minimize thecomplexity of manufacturing, e.g., to reduce the number of printingsteps. Preferably, they both comprise a carbon ink overlayer over asilver ink underlayer; the carbon ink providing the active electrodesurface and the silver ink providing sufficient conductivity so that,during use of the plate in an assay, electrical potential is evenlydistributed throughout a particular section. When forming such layers,e.g., by a two step printing process, it is beneficial that theoverlayer be of slightly larger dimensions than the underlayer and thatit be of suitable thickness to ensure that a sample in wells 1002 is notexposed to the underlayer material. It may be beneficial to print ordeposit the overlayer in multiple layers so as to ensure that theunderlayer is completely covered so that the underlayer does notinterfere with subsequent processing steps or with ECL measurements(e.g., a preferred electrode material comprises three layers of carbonink over a layer of silver ink, the layers most preferably beingdeposited by screen printing). Dielectric layer 1040 is an electricallyinsulating film, preferably formed from a dielectric ink by a printingprocess such as screen printing. Dielectric layer 1040 is patterned soas to define the surfaces of conductive layer 1050 that contact fluidsin wells 1002 (i.e., the surfaces that are not covered). Holes 1042 indielectric layer 1040 define fluid containment regions on the workingelectrode sections 1052 of conductive layer 1050. In such fluidcontainment regions, the dielectric layer acts as a barrier that can beused to confine small volumes of fluids over the working electrode,e.g., to aid in depositing assay reagents onto selected assay domainswithin a well. Holes 1042 in dielectric layer 1040 define one fluidcontainment regions and/or assay domains on the working electrodesurface within each well of plate 1000. Optionally, dielectric layer1040 may be omitted (in such a case, reagents may still be depositedinto defined assay domains by controlled deposition, e.g., usingmicrodispensing or pin transfer techniques).

Contact layer 1070 is a conductive layer that allows for electricalconnection of the multi-well assay plate to an external source ofelectrical energy. The contact layer is sectioned in a series of workingelectrode contacts 1072 and counter electrode contacts 1074 to allowindependent connection to specific sections of electrodes 1052 and 1054.The contact layers are, preferably, formed by printing, most preferablyscreen printing, a silver ink under layer (to provide high conductivity)followed by a carbon ink overlayer (to prevent corrosion of the silverink and prevent any deleterious effects by the exposed silver on asubsequent plasma processing step). Holes 1062 and 1064 in substrate1060 are, preferably, made by a cutting process such as die cutting orlaser drilling. Holes 1062 are filled with a conductive material toprovide an electrical connection between working electrode contacts 1072and working electrode sections 1052. Holes 1064 are filled withconductive material to provide an electrical connection between counterelectrode contacts 1074 and counter electrode sections 1054. Holes 1062and 1064 are preferably filled with conductive material during theformation of conductive layer 1050 or contact layer 1070, e.g., duringthe printing of a conductive ink on a substrate, excess ink is forcedinto holes in the substrate so as to fill the holes with the conductiveink.

In operation, test samples are introduced into wells of plate 1000. Asource of electrical energy is connected across one or more workingelectrode sections 1052 and one or more counter electrode sections 1054(via one or more of working electrode contacts 1072 and one or more ofcounter electrode contacts 1074, respectively). Application ofelectrical energy across these connections leads to the application ofan electrochemical potential across the test samples via the exposedsurfaces of electrode sections 1052 and 1054 (the application ofelectrochemical potential being confined to wells in sectors contactingworking electrode and counter electrode sections that are in electricalconnection to the source of electrical energy).

The structure shown in FIGS. 10A and 10B is readily modified so as to beapplicable to plates having different numbers of wells, differentarrangements of wells and/or different arrangements of independentlyaddressable sectors. Preferred embodiments include 96-well plates having4, 7, or 10 assay domains per well and 24-well plates having 25, 64 or100 wells per plate. FIG. 10C shows dielectric layer 1140, amodification of dielectric layer 1040 designed to expose 4 “fluidcontainment regions” 1141 on the working electrode surface of each well(the figure is only shown for one sector of the plate). FIG. 10D shows astylized cross-sectional view of 3 wells of plate 1100 which isidentical to plate 1000 except for the replacement of dielectric layer1040 with dielectric layer 1140.

FIG. 11 shows, multi-well assay plate 1500, an embodiment of theinvention that is particularly well suited for genomic or proteomicanalysis. The size of the wells is chosen so as to optimize theefficiency of the imaging of luminescence generated from the wells bythe imaging instrument (as described below). Multi-well assay plate 1500is a laminar structure comprising, in sequence, plate top 1520, adhesivelayer 1530, conductive tape layer 1514B, dielectric layer 1540,conductive layer 1552, substrate 1560, contact layer 1572 and conductivetape layer 1514A. Element 1580 shows layers 1572, 1560, 1552 and 1540aligned and stacked, in order from top to bottom, 1540 (top), 1552,1560, 1572 (bottom). Conductive tape layers 1514A and 1514B are providedby folding conductive tape 1510 around element 1580 at fold 1516. Holes1522, 1532 and 1518 are aligned so as to form a plurality of wellshaving well bottoms defined by element 1580. Through-holes 1562 throughsubstrate 1560 provide an electrical path between conductive layer 1552and contact layer 1572. Through holes 1512 through conductive tape layer1514A provide access to contact layer 1572 (and, therefore a way tocontact conductive layer 1552). Plate top 1520 is analogous to plate top1020 from FIG. 10 except for the specific arrangement of wells. Adhesivelayer 1530 is an adhesive analogous to adhesive layer 1030 in FIG. 10and may be omitted. Conductive tape 1510 is a laminate structurecomprising a conductive film on an insulating and adhesive substrate(preferably, a plastic film coated on one side with an evaporated layerof aluminum and on the other side with an adhesive). Substrate 1560,conductive layer 1552, dielectric layer 1540 and contact layer 1572 aresimilar in composition and preparation to substrate 1060, conductivelayer 1050, dielectric layer 1040 and contact layer 1072 as describedfor FIG. 10. Conductive layer 1552 is sectioned into 6 square sectionsso as to divide plate 1500 into 6 independently addressable sectors(each having one well). Holes 1542 through dielectric layer 1540, definea large number (preferably 10-50,000, more preferably 100-10,000; 256are shown in the figure) of fluid containment regions in each well.Binding reagents such as specific nucleic acid sequences or specificproteins can be selectively introduced and or immobilized into specificfluid containment regions by selectively microdispensing the bindingreagents into the specific fluid containment regions.

While the figures illustrating embodiments of the plates of theinvention have shown specific patterns for number, shape anddistribution of wells, sectors and fluid containment regions/assaydomains, it should be clear that the designs are adaptable so as toallow for a wide variation in these parameters.

The assay domains and immobilized layers of the invention are useful forcarrying out a wide variety of established assay formats, e.g., assaysbased on the measurement of electrochemical voltage and/or current or,preferably, an electrode-induced luminescence, most preferably,electrochemiluminescence. For examples of methods for conducting ECLassays, the reader is directed towards U.S. Pat. Nos. 5,591,581;5,641,623; 5,643,713; 5,705,402; 6,066,448; 6,165,708; 6,207,369; and6,214,552 and Published PCT Applications WO87/06706 and WO98/12539,these references hereby incorporated by reference. Assays may bedirected to, but are not limited to, the measurement of the quantity ofan analyte; the measurement of a property of a sample (e.g.,temperature, luminescence, electrochemical activity, color, turbidity,etc.); the measurement of a chemical, biochemical and/or biologicalactivity (e.g., an enzymatic activity); the measurement of a kinetic orthermodynamic parameter (e.g., the rate or equilibrium constant for areaction), etc.

The embodiments of the invention can be used to test a variety ofsamples which may contain an analyte or activity of interest. Suchsamples may be in solid, emulsion, suspension, liquid, or gas form. Theymay be, but are not limited to, samples containing or derived from, forexample, cells (live or dead) and cell-derived products, cell fragments,cell fractions, cell lysates, organelles, cell membranes, cell culturesupernatants (including supernatants from antibody producing organismssuch as hybridomas), waste or drinking water, food, beverages,pharmaceutical compositions, blood, serum, plasma, hair, sweat, urine,feces, tissue, saliva, mucous, oils, sewage, environmental samples,organic solvents or air. The sample may further comprise, for example,water, organic solvents (e.g., acetonitrile, dimethyl sulfoxide,dimethyl formamide, n-methyl-pyrrolidone or alcohols) or mixturesthereof.

Analytes that may be measured include, but are not limited to, wholecells, cell surface antigens, subcellular particles (e.g., organelles ormembrane fragments), viruses, prions, dust mites or fragments thereof,viroids, antibodies, antigens, haptens, fatty acids, nucleic acids (andsynthetic analogs), proteins (and synthetic analogs), lipoproteins,polysaccharides, inhibitors, cofactors, haptens, cell receptors,receptor ligands, lipopolysaccharides, glycoproteins, peptides,polypeptides, enzymes, enzyme substrates, enzyme products, secondmessengers, cellular metabolites, hormones, pharmacological agents,synthetic organic molecules, organometallic molecules, tranquilizers,barbiturates, alkaloids, steroids, vitamins, amino acids, sugars,lectins, recombinant or derived proteins, biotin, avidin, streptavidin,or inorganic molecules present in the sample. Activities that may bemeasured include, but are not limited to, the activities ofphosphorylases, phosphatases, esterases, trans-glutaminases, nucleicacid damaging activities, transferases, oxidases, reductases,dehydrogenases, glycosidases, ribosomes, protein processing enzymes(e.g., proteases, kinases, protein phosphatases, ubiquitin-proteinligases, etc.), nucleic acid processing enzymes (e.g., polymerases,nucleases, integrases, ligases, helicases, telomerases, etc.), cellularreceptor activation, second messenger system activation, etc.

In one embodiment of the invention, a sample potentially containing aluminescent, chemiluminescent and/or redox-active substance (preferablyan ECL-active substance) is introduced to an assay plate or one or morewells of an assay plate of the invention and an electrochemical orluminescent signal (preferably, electrochemiluminescence) from thesample is induced and measured from one or more assay domains so as tomeasure the quantity of the substance and/or identify the substance. Inanother embodiment of the invention, a sample containing a luminescent,chemiluminescent and/or redox-active substance (preferably an ECL-activesubstance) is introduced to an assay plate or one or more wells of anassay plate of the invention and an electrochemical or luminescentsignal (preferably, electrochemiluminescence) from the sample is inducedand measured from one or more assay domains so as to measure thepresence of substances, chemical activities or biological activitiesthat affect the production of the signal from the substance (e.g., thepresence, production and/or consumption of ECL coreactants, hydrogenions, luminescence quenchers, chemiluminescence triggers, etc.). Inother embodiments of the invention, luminescent, chemiluminescent and/orredox-active substances (preferably ECL-active substances) are used aslabels to allow the monitoring of assay reagents such as enzymesubstrates or binding reagents. Assay formats for measuring analytesthrough the use of labeled binding reagents specific for the analyteinclude homogeneous and heterogeneous methods. Heterogeneous methods mayinclude a wash step to separate labels (and/or labels attached to amaterial) on a solid phase/electrode from labels in solution.

A wide variety of materials have been shown to emit electrode inducedluminescence, particularly electrochemiluminescence, and may be usedwith the methods, plates, kits, systems and instruments of theinvention. In preferred electrochemiluminescence systems, ECL-activematerials and/or labels are regenerated after the emission ofelectrochemiluminescence and, during an electrochemiluminescenceexperiment, may be repeatedly excited to an excited state and/or inducedto emit luminescence. For example, one class of ECL-active materials arebelieved to function via a mechanism that includes the steps of i)oxidation of the material; ii) reduction of the oxidized material by astrong reducing agent so as to produce the material in an excited stateand iii) emission of a photon from the excited state so as to regeneratethe ECL-active material. Preferably, the difference in redox potentialsbetween the ECL-active material and the strong reducing agent issufficient so that the energy released by step (ii) is equal to orgreater than the energy of the photon. In an analogous mechanism, steps(i) and (ii) may be replaced by i) reduction of the material and ii)oxidation of the reduced material by a strong oxidizing agent. In someespecially preferred systems, the mechanism is believed to furthercomprise the step of oxidizing an ECL coreactant so as to form thestrong reducing agent or, analogously, reducing an ECL coreactant toform the strong oxidizing agent.

Preferred luminescent materials and labels include luminescentorganometallic complexes of Ru, Os and Re. Some especially usefulmaterials are polypyridyl complexes of ruthenium and osmium, inparticular, complexes having the structure ML¹L²L³ where M is rutheniumor osmium, and L¹, L² and L³ each are bipyridine, phenanthroline,substituted bipyridine and/or substituted phenanthroline. We have foundthat the inclusion of substituted bipyridines or phenanthrolinespresenting substituents comprising negatively charged groups, preferablysulfate groups and most preferably sulfonate groups (as described incopending U.S. patent application Ser. No. 09/896,974, entitled “ECLLabels Having Improved Non-Specific Binding Properties, Methods of Usingand Kits Containing the Same” filed Jun. 29, 2001, the disclosure herebyincorporated by reference) are especially preferred due to theirresistance to non-specific binding, in particular to electrodescomprising carbon, carbon particles, carbon fibrils, carbon composites,carbon fibril composites and/or carbon inks.

The invention also relates to detection methods using the electrodes ofthe present invention.

One aspect of the invention relates to methods of measuring an analyteof interest, wherein the analyte of interest is immobilized on anelectrode (preferably in an assay domain of an assay cell or assaywell). One embodiment comprises the steps of: i) immobilizing theanalyte of interest on an electrode, preferably within an assay domain,e.g., by contacting the electrode with a sample comprising the analyteof interest and ii) measuring the analyte of interest. Theimmobilization preferably proceeds via the formation of covalent bondsto functional groups on the electrode, or more preferably via theformation of specific binding interactions with binding reagentsimmobilized on the electrode, or most preferably via passive adsorptionon the electrode.

Another aspect of the invention relates to methods of measuring ananalyte of interest that binds to a biomaterial, wherein the biomaterialis immobilized on an electrode (preferably in an assay domain of anassay cell or assay well). One embodiment comprises the steps of i)contacting the biomaterial with a sample comprising the analyte; ii)forming a complex on the electrode comprising the analyte and thebiomaterial and ii) measuring the analyte of interest. The biomaterialis preferably immobilized on the electrode via covalent bonds tofunctional groups on the electrode, or more preferably via specificbinding interactions with a capture reagent immobilized on theelectrode, or most preferably via passive adsorption on the electrode.Optionally, the assay method also comprises the step of immobilizing thebiomaterial on the electrode. This immobilization step can be carriedout before, during and/or after the step of contacting the biomaterialwith the sample.

Preferably, the aforementioned methods of measuring an analyte furthercomprise the steps of applying electrical energy (e.g., current orvoltage) to the electrode (preferably, under conditions appropriate forinducing electrochemiluminescence, e.g., in the presence of an ECLcoreactant) and measuring luminescence (preferably,electrochemiluminescence) induced at the electrode (e.g., from aluminescent species, preferably an electrochemiluminescent species,associated with the analyte), wherein the luminescence signal correlatesto the amount of analyte present. Optionally, the method may comprisethe step of introducing an ECL coreactant prior to the induction ofluminescence. The luminescent species may be the analyte itself or itmay be a luminescent species linked to the analyte. Such linkages mayinclude i) a covalent bond, ii) a specific binding interaction (e.g.,via a labeled antibody directed against the analyte) and/or iii) anon-specific binding interaction. The assay method, preferably, furthercomprises the step of forming the linkage between the label and theanalyte, e.g., by contacting or mixing the analyte with a label or alabeled reagent such as a labeled binding reagent. The formation of thelinkage may be carried out before, during and/or after theimmobilization step. The assay method may also include one or more washsteps to remove material (e.g., analyte, biomaterial, blocking reagent,labeled reagent, etc.) that is not bound to the electrode.

Another aspect of the invention relates to methods of measuring abinding interaction of a biomaterial with a binding partner, wherein thebiomaterial is immobilized on an electrode (preferably in an assaydomain of an assay cell or assay well). One embodiment comprises thesteps of i) contacting the biomaterial with a binding partner of thebiomaterial; ii) forming a complex on the electrode comprising thebiomaterial and the binding partner and ii) measuring the complex so asto measure the binding interaction. The biomaterial is preferablyimmobilized on the electrode via covalent bonds to functional groups onthe electrode, or more preferably via specific binding interactions witha capture reagent immobilized on the electrode, or most preferably viapassive adsorption on the electrode. Optionally, the assay method alsocomprises the step of immobilizing the biomaterial on the electrode.This immobilization step can be carried out before, during and/or afterthe step of contacting the biomaterial with the binding partner. Themeasurement of the binding interaction may be used in a variety ofapplications including, but not limited to, i) measuring the amount ofthe biomaterial; ii) measuring the amount of the binding partner andiii) measuring the affinity of a biomaterial for binding partner. Theassay method may further comprise the step of contacting the biomaterialand/or the binding partner with an inhibitor of the binding interactionso that the extent of binding is indicative, e.g., of the amount of theinhibitor or the inhibition constant of the inhibitor. The inhibitionassay may also be used to screen compounds for inhibitors of the bindinginteraction.

Preferably, the aforementioned method of measuring a binding interactionfurther comprises the steps of applying electrical energy (e.g., currentor voltage) to the electrode (preferably, under conditions appropriatefor inducing electrochemiluminescence, e.g., in the presence of an ECLcoreactant) and measuring luminescence (preferably,electrochemiluminescence) induced at the electrode (e.g., from aluminescent species, preferably an electrochemiluminescent species,associated with the binding partner), wherein the luminescence signalcorrelates to the number of binding interactions. Optionally, the methodmay comprise the step of introducing an ECL coreactant prior to theinduction of luminescence. The luminescent species may be the bindingpartner itself or it may be a luminescent species linked to the bindingpartner. Such linkages may include i) a covalent bond, ii) a specificbinding interaction (e.g., via a labeled antibody directed against thebinding partner) and/or iii) a non-specific binding interaction. Theassay method, preferably, further comprises the step of forming thelinkage between the label and the binding partner, e.g., by contactingor mixing the binding partner with a label or a labeled reagent such asa labeled binding reagent. The formation of the linkage may be carriedout before, during and/or after the immobilization step. The assaymethod may also include one or more wash steps to remove material (e.g.,binding partner, biomaterial, blocking reagent, labeled reagent, etc.)that is not bound to the electrode.

Another aspect of the invention relates to methods of measuring anactivity or process that modifies a substance, the method comprising thesteps of subjecting the substance to a sample comprising the activity orto conditions under which the process occurs and measuring the extent ofthe modification so as to measure the activity or process. The extent ofthe modification is, preferably, measured by selectively measuring themodified substance and/or the remaining unmodified substance accordingto the assay methods of the invention (e.g., by using labeled antibodiesspecific for the starting material or product). Optionally, the activityor process is carried out in the presence of an inhibitor of theactivity or process so that the extent of modification is indicative,e.g., of the amount of the inhibitor or the inhibition constant of theinhibitor. The inhibition assay may also be used to screen compounds forinhibitors of the binding interaction and/or for measuring an activityor process that modifies a binding partner of an immobilized substance.

In one embodiment, a substance is immobilized on an electrode(preferably in an assay domain of an assay cell or assay well) andsubjected to a modifying activity or process, and assayed to determinethe extent of modification. In another embodiment, a substance issubjected to a modifying activity or process, immobilized on anelectrode, and assayed to determine the extent of modification. In yetanother embodiment, a cell is subjected to a modifying activity orprocess, the cell is lysed, a biological membrane or other componentderived from the cell (e.g., an protein, nucleic acid, second messenger,organelle, membrane fragment, membrane vesicle, membrane ghost, membraneprotein, membrane lipid, etc) is immobilized on an electrode, andassayed to determine the extent of modification. Examples of activitiesand processes that can be measured include kinaseactivity/phosphorylation (including autophosphorylation of membranebound kinases), phosphatase activity/dephosphorylation, changes inmembrane lipid composition or orientation (e.g., changes in phosphatidylserine levels during apoptosis), hydrolysis or changes inphosphorylation state of membrane phosphatidyl inositols, prenylation ormyristoylation of proteins, binding and/or release of soluble proteinsand/or peripheral membrane proteins to biological membranes, transfer ofproteins and/or lipids between biological membranes (e.g., betweenorganelles and/or between an organelle and the cytoplasmic membrane),etc.

One embodiment of the method of measuring an activity or process (or,alternatively, an inhibitor of an activity or process) that modifies asubstance relates to measuring an activity or process that results fromthe activation of a membrane protein (e.g., as a result of a change inthe physical or chemical environment, a change in membrane potential,the aggregation of the protein, the binding of a ligand to a membranereceptor, etc.). For example, the activation of a membrane protein maylead to phosphorylation of the protein or of other components of themembrane (the phosphorylated components being measured, e.g., usingphosphopeptide specific antibodies); ii) the sequestration or binding(or, alternatively, the release) to the membrane of soluble cellularcomponents such as peripheral membrane proteins or cytoplasmic proteins(the binding of soluble cellular components being measured, e.g., usingantibodies specific for the components); iii) the up or down regulationof membrane proteins (the membrane proteins being measured, e.g., usingantibodies specific for the specific membrane protein being monitored),etc.

Another aspect of the invention relates to kits for use in conductingassays, preferably luminescence assays, more preferably electrodeinduced luminescence assays, and most preferablyelectrochemiluminescence assays, comprising an assay module, preferablyan assay plate, more preferably a multi-well assay plate, and at leastone assay component selected from the group consisting of bindingreagents, enzymes, enzyme substrates and other reagents useful incarrying out an assay. Examples include, but are not limited to, wholecells, cell surface antigens, subcellular particles (e.g., organelles ormembrane fragments), viruses, prions, dust mites or fragments thereof,viroids, antibodies, antigens, haptens, fatty acids, nucleic acids (andsynthetic analogs), proteins (and synthetic analogs), lipoproteins,polysaccharides, lipopolysaccharides, glycoproteins, peptides,polypeptides, enzymes (e.g., phosphorylases, phosphatases, esterases,trans-glutaminases, transferases, oxidases, reductases, dehydrogenases,glycosidases, protein processing enzymes (e.g., proteases, kinases,protein phosphatases, ubiquitin-protein ligases, etc.), nucleic acidprocessing enzymes (e.g., polymerases, nucleases, integrases, ligases,helicases, telomerases, etc.)), enzyme substrates (e.g., substrates ofthe enzymes listed above), second messengers, cellular metabolites,hormones, pharmacological agents, tranquilizers, barbiturates,alkaloids, steroids, vitamins, amino acids, sugars, lectins, recombinantor derived proteins, biotin, avidin, streptavidin, luminescent labels(preferably electrochemiluminescent labels), electrochemiluminescencecoreactants, pH buffers, blocking agents, preservatives, stabilizingagents, detergents, dessicants, hygroscopic agents, etc. Such assayreagents may be unlabeled or labeled (preferably with a luminescentlabel, most preferably with an electrochemiluminescent label). Oneembodiment of the invention includes a kit for use in conducting assays,preferably luminescence assays, more preferably electrode inducedluminescence assays, and most preferably electrochemiluminescenceassays, comprising an assay module, preferably an assay plate, morepreferably a multi-well assay plate, and at least one assay componentselected from the group consisting of: (a) at least one luminescentlabel (preferably electrochemiluminescent label); (b) at least oneelectrochemiluminescence coreactant); (c) one or more binding reagents;(d) a pH buffer; (e) one or more blocking reagents; (f) preservatives;(g) stabilizing agents; (h) enzymes; (i) detergents; (j) desiccants and(k) hygroscopic agents.

Preferably, the kit comprises the assay module, preferably an assayplate, and the assay component(s) in one or more, preferably two ormore, more preferably three or more containers.

Preferably, the assay module is a multi-well plate is adapted for use inconducting the electrode induced luminescence assays (preferablyelectrochemiluminescence assays) in sectors.

According to one embodiment, the kit comprises one or more of the assaycomponents in one or more plate wells, preferably in dry form.

According to one embodiment, the assay components are in separatecontainers. According to another embodiment, the kit includes acontainer comprising binding reagents and stabilizing agents. Accordingto another embodiment, the well reagents may include binding reagents,stabilizing agents. Preferably, the kits do not contain any liquids inthe wells.

One preferred embodiment relates to a kit for use in conductingelectrode induced luminescence assays (preferablyelectrochemiluminescence assays) comprising an assay plate, preferably amulti-well assay plate, and at least one assay component selected fromthe group consisting of at least one luminescent label (preferablyelectrochemiluminescent label) and at least one electrochemiluminescencecoreactant).

Another embodiment relates to a kit comprising a multi-well plate and atleast one electrode induced luminescent label (preferablyelectrochemiluminescent label) and/or at least one bioreagent and/or atleast one blocking reagent (e.g., BSA).

According to one preferred embodiment, the kit comprises at least onebioreagent, preferably immobilized on the plate surface selected from:antibodies, fragments of antibodies, proteins, enzymes, enzymesubstrates, inhibitors, cofactors, antigens, haptens, lipoproteins,liposaccharides, cells, sub-cellular components, cell receptors,viruses, nucleic acids, antigens, lipids, glycoproteins, carbohydrates,peptides, amino acids, hormones, protein-binding ligands,pharmacological agents, luminescent labels (preferably ECL labels) orcombinations thereof.

According to another preferred embodiment, the kit comprises at leastone biological membrane or component thereof, preferably immobilized onthe plate surface, that comprises an active protein selected from:single transmembrane receptors with intrinsic tyrosine kinase activity;non-tyrosine kinase transmembrane receptors (e.g., transferrinreceptor); G-protein coupled receptors (GPCR); GPCR effector proteins(e.g., adenylate cyclase); phosphoinositides (e.g., phosphatidy inositol4,5 bisphosphate (PIP₂)); phospholipid or sphingolipid composition,identification, or function (i.e., changes in phosphotidylserinepresence during apoptosis); organelle-bound GTPases/guanine nucleotideexchange factors (GEFs)/GTPase activating proteins (GAPs);cytokine/chemokine receptors; cell adhesion molecules (e.g., VCAM,integrins); cytoplasmic peripheral membrane protein kinases (e.g., src);intracellular protein kinase adaptor/docking proteins (e.g., insulinreceptor substrate 1, GRB2); ion channels (e.g., nicotinic acetylcholinereceptor, CFTR, etc.); passive transporters (e.g., glucose); active(ATP-driven) transporters; ion-linked transporters (e.g., Na+/glucose);glycosyltranferases/glycoprotein modifying enzymes; nuclear membranefragments; and soluble receptors.

Preferably, the kit includes immobilized reagents comprised of proteins,nucleic acids, or combinations thereof.

According to one preferred embodiment, the plurality of wells includesat least two different bioreagents. For example, a well may include twoor more assay domains, wherein two or more assay domains have differentbioreagents.

Preferably, the kit comprises at least one electrochemiluminescencecoreactant and/or at least one electrode induced luminescence label(preferably electrochemiluminescent label).

Another aspect of the invention relates to improved methods and systemsfor selecting or identifying biologically active compounds and,optionally, incorporating such biologically active compounds intosuitable carrier compositions in appropriate dosages. The inventionincludes the use of the assay electrodes, kits and/or methods of theinvention to screen for new drugs, preferably, by high-throughputscreening (HTS), preferably involving screening of greater than 50, morepreferably 100, more preferably 500, even more preferably 1,000, andmost preferably 5,000. According to a particularly preferred embodiment,the screening involves greater than 10,000, greater than 50,000, greaterthan 100,00, greater than 500,000 and/or greater than 1,000,000compounds.

One embodiment of the invention relates to a method for selecting oridentifying biologically active compounds from a library of compounds,said method comprising screening said library of compounds forbiological or biochemical activity, wherein said screening includesassaying the library of compounds for the biological or biochemicalactivity, the assays being conducted using the assay electrodes of theinvention.

Preferably, the method further comprises identifying one or more activecompounds.

Preferably, the method further comprises testing said one or more activecompounds for bioavailability, toxicity and/or biological activity invivo. According to one preferred embodiment, the testing comprisesfurther screening using the assay electrodes of the invention.

Preferably, the method further comprises synthesizing analogues of saidone or more active compounds. According to one preferred embodiment, theanalogues are screened for bioavailability, biological activity and/ortoxicity using the assay electrodes of the invention.

According to a particularly preferred embodiment, the method furthercomprises formulating the one or more compounds into drugs foradministrating to humans and/or animals.

Preferably, the formulating comprises determining the suitable amount ofthe one or more active compounds in the drug and mixing the suitableamount with one or excipients or carriers. Preferably, the excipientcomprises sugar and/or starch.

Another embodiment of the invention relates to a method of analyzing oneor more complex mixtures of biochemical substances to measure aplurality of binding components therein, comprising:

(a) contacting said mixtures with one or more assay electrodes havingone or more lipid/protein layers immobilized thereon, preferably byadding said mixtures to a multi-well plate adapted for electrode inducedluminescence assays (preferably electrochemiluminescence assays),wherein the wells of the plate include the assay electrodes;

(b) applying a voltage or current to the electrodes sufficient to induceluminescence; and

(c) measuring emitted luminescence.

Another embodiment of the invention relates to a method of analyzing theoutput of one or more combinatorial (biological and/or chemical)mixtures to measure a plurality of binding components therein,comprising:

(a) contacting said mixtures to one or more assay electrodes, preferablyby introducing said mixture into a multi-well plate adapted forelectrode induced luminescence (preferably electrochemiluminescence)assays, said plate having a plurality of wells comprising one or moreassay electrodes;

(b) applying a voltage or current to the electrodes sufficient to induceluminescence; and

(c) measuring emitted luminescence.

Another embodiment of the invention relates to a method for measuring asingle biochemical substance in a sample in a multiplicity ofsimultaneous assays, comprising:

(a) contacting said sample with an assay electrode, preferably byintroducing said sample into a multi-well plate adapted for electrodeinduced luminescence (preferably electrochemiluminescence) assays, saidplate having a plurality of wells comprising one or more assayelectrodes;

(b) applying a voltage or current to the electrodes sufficient to induceluminescence; and

(c) measuring emitted luminescence.

A further embodiment of the invention relates to a method of drugdiscovery comprising:

(a) selecting a multiplicity of compounds for testing;

(b) screening said multiplicity of compounds for biological activity(using any one of the multi-well plates and/or apparatus describedabove) to find one or more biologically active compounds; and

(c) modifying said one or more biologically active compounds to reducetoxicity and/or enhance biological activity thereby forming one or moremodified biologically active compounds.

Preferably, the method further comprises screening said modifiedbiologically active compounds for biological activity and/or toxicity(using the assay electrodes of the invention described above).

Preferably, the method further comprises determining the appropriatedosage of one or more of said modified biologically active compounds.Preferably, the method still further comprises incorporating such dosageinto a suitable carrier such as sugar or starch to form a drug in solid(e.g., pill or tablet) or liquid form.

Advantageously, the assay electrodes, assay modules and methods of theinvention may be integrated into and/or used in a variety of screeningand/or drug discovery methods. Such screening and/or drug discoverymethods include those set forth in U.S. Pat. No. 5,565,325 to Blake;U.S. Pat. No. 5,593,135 to Chen et al.; U.S. Pat. No. 5,521,135 toThastrup et al.; U.S. Pat. No. 5,684,711 to Agrafiotis et al.; U.S. Pat.No. 5,639,603 to Dower et al.; U.S. Pat. No. 5,569,588 to Ashby et al.;U.S. Pat. No. 5,541,061; U.S. Pat. No. 5,574,656; and U.S. Pat. No.5,783,431 to Peterson et al.

According to another embodiment, the invention further comprisesidentifying adverse effects associated with the drug and storinginformation relating to the adverse effects in a database. See, U.S.Pat. No. 6,219,674 by Classen, hereby incorporated by reference.

Another aspect of the invention relates to improved biologically activecompounds and/or drugs made using the inventive methods.

EXAMPLES

The following examples are illustrative of some of the electrodes,plates, kits and methods falling within the scope of the presentinvention. They are, of course, not to be considered in any waylimitative of the invention. Numerous changes and modification can bemade with respect to the invention by one of ordinary skill in the artwithout undue experimentation.

Example 1 Fabrication of Multi-Well Assay Plates having Screen PrintedElectrodes

Multi-layer plate bottoms were prepared by screen printing electrodesand electrical contacts on 0.007″ thick Mylar polyester sheet. The Mylarsheet was first cut with a CO₂ laser so to form conductive through-holes(i.e., holes that were subsequently made conductive by filling withconductive ink) as well as to form alignment holes that were used toalign the plate bottom with the plate top. Electrical contacts wereformed on the bottom of the Mylar sheet by screen printing anappropriately patterned silver ink layer (Acheson 479ss) and a carbonink overlayer (Acheson 407c). The carbon ink layer was dimensionedslightly larger (0.01 inches) than the silver ink layer to preventexposure of the edge of the silver film. Working and counter electrodeswere formed on the top of the Mylar film in a similar fashion exceptthat three layers of carbon ink were used to ensure that no silverremained exposed. The conductive through-holes filled with conductiveink during these screen-printing steps. A dielectric ink wassubsequently printed over the electrode layers so as to define theactive exposed surface area of the working electrode. Typically, nineplate bottoms were simultaneously printed on an 18″×12″ Mylar sheet.Typical registrational tolerances during the screen printing steps were+/−0.007-0.008 inches on the top side of the substrate and +/−0.010inches on the bottom side. The separation between the printed counterand working electrode strips was kept at >0.010 inches to prevent theformation of short circuits. Optionally, the working electrodes wereconditioned by treating the patterned plate bottoms for 5 min. with anoxygen plasma (2000 W, 200 mtorr) in a plasma chamber (Series B,Advanced Plasma Systems, St. Petersburg, Fla.) modified with large areaflat electrodes.

Multi-well assay plates were assembled using the plate bottoms describedabove and injection molded plate tops. The dimensions of the plate topsmet industry standards as established by the Society of BiomolecularScreening. The plate tops were either made of black plastic (polystyreneloaded with black pigment) or white plastic (polystyrene loaded withtitanium dioxide). The bottom surfaces of the plate tops were contactedwith die-cut double sided tape (1 mil PET coated on each side with 2 milof acrylic pressure sensitive adhesive) so as to allow for sealing ofthe plate tops to the plate bottoms. The tape was cut to form holes thatwere slightly oversized relative to the holes in the plate tops. Theplate bottoms were fixed (using the laser cut alignment holes) ontoalignment pins on an X-Y table. The plate bottoms were optically alignedto the plate tops and then sealed together using a pneumatic press (400pounds, 10 s). Alignment was carried out sufficiently accurately so thatthe exposed working electrodes were centered within the wells (+/−0.020inches for 96-well plates and +/−0.015 inches for 384 well plates).These tolerances ensured that the exposed regions of the workingelectrodes were within the wells and that there were exposed counterelectrode surfaces on both sides of the working electrode. In someexamples, assay reagents were deposited and dried on the plate bottomsprior to assembly of the plate.

A variety of types of multi-well assay plates were prepared according tothe procedure described above. A few specific plate designs aredescribed in more detail below to allow for reference in subsequentexamples. Plate B, a 96-well plate sectioned into 6 square sectors of4×4 wells, was prepared using components and patterns as pictured inFIG. 10A and had a black plate top. Plate C, a 96-well plate sectionedinto 6 square sectors of 4×4 wells, was prepared using components andpatterns as pictured in FIG. 10 (except that the dielectric layer inPlate C is patterned so as to expose four isolated “fluid containmentregions” on the working electrode surface within each well, see FIGS.10C and 10D) and a black plate top. Plate D was similar to Plate Cexcept that the dielectric layer was patterned so as to expose 7isolated “fluid containment regions” on the working electrode withineach well. Plate E was similar to Plate C except that the dielectriclayer was patterned so as to expose 10 isolated “fluid containmentregions” on the working electrode within each well. Plate F, G and Hwere analogous to Plate C, except that they had 24 wells that weresquare in shape (the plates being sectored into 6 square sectors of 4wells) and the dielectric layer was patterned to expose 25, 64 or 100fluid containment regions, respectively. In FIG. 10A, details A, B, Cand D show for Plate B: the printed contact layer, the Mylar film withthrough-holes, the printed electrode layer and the printed dielectriclayer (in one sector of the plate) , respectively.

Example 2 ECL Measurements

Plates were read on an instrument designed to make electrical contact toindividual square sectors. The sector in electrical contact with theinstrument was aligned with a telecentric lens (having a front elementwith a diameter of 4.1″) coupled to a cooled CCD camera (VersArray:1300F, Princeton Instruments) that was used to image ECL emitted fromthe sector. The camera employed a CCD chip with dimensions of roughly2.6 cm×2.6 cm and having a 1340×1300 array of pixels. The pixel size was0.02 mm×0.022 mm. An optical band pass filter in the optical path wasused to select for light matching the emission profile ofruthenium-tris-bipyridine. A translation table was used to translate theplate under the telecentric lens so as to allow all 6 sectors to beread. Image analysis software was used to identify wells or assaydomains within wells and to quantitate ECL from specific wells ordomains. ECL from plates having screen printed carbon working electrodeswas induced using a linear voltage scan from roughly 2-5 V over 3seconds unless otherwise indicated. ECL is reported as the totalintegrated light signal measured over the period of the voltage scan(after correcting for background light levels and detector offset).

Example 3 An ECL Assay Measuring Multiple Activities of an Enzyme in OneWell of an MDMW Plate

Many nucleic acid processing enzymes have both nucleic acid synthesizing(e.g., polymerase or ligase) activities and nuclease activities. Oneexample is HIV Reverse Transcriptase (RT), an enzyme with both aRNA-dependent DNA polymerase (RDDP) activity and an RNAse H activity.The following example demonstrates an ECL assay for measuring both HIVRT activities in one well of an MDMW Plate.

The assay format is illustrated in FIG. 12. The enzyme substrate is a5′-labeled (using TAG phosphoramidite, IGEN International, Inc.) DNAprimer bound to the 3′-end of an RNA target sequence. The RDDP activityextends the DNA primer to make a complementary copy of the RNA sequence.The RNAse H activity selectively hydrolyzes RNA in RNA-DNA duplexes.RNAse activity is measured by hybridizing the labeled DNA product to animmobilized probe (3′-B13) that is complementary to the DNA primersequence (thus measuring the RNAse catalyzed exposure of the DNAprimer). The RDDP activity is measured by hybridizing the labeled DNAproduct to an immobilized probe (5′-B13) that is complementary to theextended DNA sequence (thus measuring the RDDP extension of the DNAprimer).

The assay was conducted on a MDMW plate adapted for electrode inducedchemiluminescence measurements and having four fluid containment regionsexposed on the working electrode surface of each well (Plate C ofExample 1). The two probes (3′-B13 and 5′-B13) were biotin-labeled tofacilitate immobilization. Each probe was pre-bound to avidin. Assaydomains were formed by immobilizing each probe in one fluid containmentregion of each well by microdispensing the avidin-probe complexes ontothe fluid containment regions (between 100-1000 nL containing 1 pmol ofprobe) using a non-contact microdispensor (BioDot or CartesianTechnologies) and allowing the solutions to dry. The two additionalfluid containment regions were used as control domains (one was coatedwith avidin, the other was not treated). The plates were blocked with asolution containing BSA and washed prior to use.

The assays were carried out by adding to the wells of plates 1 nmol ofthe dNTPs, 5 pmol of the substrate, 3 pmol of the enzyme and varyingamounts of an RT inhibitor in 100 uL of a buffer containing 50 mM Trisph 8.0, 40 mM KCl, 10 mM MgCl₂, 0.025% Triton® X-100, 2.5 mM DTT. Thereaction mixture was incubated for 20 min at 22° C. and then quenched bythe addition of EDTA. The plates were incubated for an additional 2hours to allow the hybridization reactions to proceed. Tripropylaminewas added (ORIGEN Assay Buffer, IGEN International and the productsassayed by electrochemiluminescence measurements. The ECL signals werecorrected by subtracting assay background (measured in wells in whichEDTA was added prior to the enzyme). FIGS. 13A-B show that the inhibitoraurintricarboxylic acid (ATA; DuPont) inhibits both RNAse H (FIG. 13B)and RDDP activities (FIG. 13A). By contrast, 125 uM ddCTP (a chainterminating agent) completely inhibited the RDDP activity but had noeffect on the RNAse H activity (data not shown).

Example 4 Detection of a Panel of Respiratory Disease Antigens

An MDMW plate adapted for ECL measurements and having 4 fluidcontainment regions on the working electrode surface exposed in eachwell (Plate C of Example 1) was coated with antibodies specific to fourrespiratory diseases: Influenza A, Influenza B, Respiratory SyncytialVirus (RSV), and Streptococcus Pyogenes (Strep A). Capture antibodysolutions (50 ug/ml in phosphate buffered saline, PBS) were dispensedusing a BioDot Dispenser onto the fluid containment regions within thewells (250 nl/spot) such that each well contained one assay domaincoated with each antibody. The solutions were allowed to dry, at whichtime a 5% BSA solution was added (200 ul/well) and the platerefrigerated overnight. The plate was washed with PBS before use (4×250ul/well).

Antigen solutions were prepared by diluting solutions of bacteria orpurified virus obtained from commercial sources by 1000× or 100×,respectively, using PBS. The approximate titers after dilution were:2.3×10¹¹ virus particles/ml Influenza A; 320 HA units/0.05 ml, or 0.1mg/ml protein for Influenza B; 6.6×10⁸ virus particles/ml RSV; 1.5×10⁴CFU/ml Strep A.

To perform the assay, 100 ul of the diluted antigen solutions werecombined with 450 ul of PBS containing 0.2% Tween-20. 75 ul of thesesolutions were combined with 10 ul of a solution of the appropriatelabeled antibody solution (sulfonated derivative of Ru(bpy)₃) such thatthe final concentration was 3 ug/ml labeled antibody. 50 ul of thissolution was added to individual wells of the washed plate and incubatedfor 8 minutes. The plate was then washed with PBS (4×200 ul/well) and100 ul of ORIGEN Assay Buffer (IGEN International) was added to eachwell. The plate was then analyzed using electrochemiluminescencedetection. FIG. 14 shows that each antigen was selectively measured inthe appropriate assay domain.

Example 5 Measurement of Tyrosine Kinase and Serine/Threonine KinaseActivities in a Well of a MDMW Plate

This example used an MDMW plate adapted for ECL measurements and having4 fluid containment regions on the working electrode surface exposed ineach well (Plate C of Example 1). Each fluid containment region received250 nL of one of the following solutions: 1 mg/ml Poly-Glu:Tyr (4:1)(PGT) in PBS buffer with 0.0075% Triton; 1 mg/ml Myelin Basic Protein(MBP) in PBS buffer with 0.0075% Triton; 0.5 mg/ml Avidin in PBS bufferwith 0.0075% Triton; 5% BSA solution in PBS. The plate was then driedovernight and blocked in a 5% BSA solution at 4° C. for 2 days. Theplate was washed to remove blocking agent prior to use.

For phosphorylation of PGT (tyrosine kinase assay) 0.1 mU/μl of c-SRCwas used, for phosphorylation of MBP (threonine kinase) 15 pg/μl ofERK-1 was used. The capture efficiency of the avidin-coated domain wasdetermined by measuring the binding of bovine IgG labeled with biotinand a sulfonated form of Ru(bpy)3.

Each spot (PGT, MBP, Avidin and BSA) was exposed to a solution of eachenzyme/analyte (as well as to mixtures of the enzymes and analytes) inthe presence of labeled (sulfonated derivative of Ru(bpy)₃) antibodiesdirected against the kinase products (anti-phosphotyrosine andanti-phospho-MBP (or, alternatively, using unlabeled primary antibodiesand labeled secondary antibodies). After incubating the plates to allowthe enzyme and binding reactions to proceed, a TPA-containing buffer wasadded and the plates were analyzed by ECL (no wash was required).Reported signals were corrected by subtracting background measured inthe absence of enzyme/analyte. Each point includes an average of fourmeasurements for background signal and 12 measurements for specificsignal. The table in FIG. 15 summarizes results of this experiment.

The PGT domain only showed high signal in the presence of the tyrosinekinase src. As expected, the MPB gave high signal in the presence of theERK-1, but also gave elevated signals in the presence of SRC, presumablybecause of the presence of several tyrosines in MPB and the relativenon-specific nature of both SRC and the anti-phophotyrosine antibody.The avidin domain gave a good signal in the presence of the biotinylatedanalyte and did not act as a substrate for the kinases. This resultdemonstrates the utility of including a binding domain, e.g., forcapturing (and, optionally, purifying) kinases to be tested from crudesamples. The BSA spot did not provide a significant signal in thepresence of the analyte/enzymes and shows that the blocking agent didnot show non-specific reactions with the assay reagents.

Example 6 Evaluation of the Detection Limits of MDMW Plates

In this Example, Applicants measured the detection limits of the ECLmeasurement of bovine IgG labeled with biotin and a sulfonatedderivative of Ru(bpy)₃ (˜2.3 labels per protein) as a function of thearea of the binding domain. Binding domains were formed by coatingAvidin onto one or more of the exposed regions (fluid containmentregions) of the electrode (by microdispensing avidin solutions anddrying on the surface of the electrode). Five plate types were prepared:

-   -   Standard 96: Plate type B from Example 1 having a single large        binding domain coated with avidin.    -   4-Spot-1: Plate type C from Example 1 having 4 small fluid        containment regions, three of which are coated with avidin to        form a binding domain.    -   4-Spot-3: Plate type C from Example 1 having 4 small fluid        containment regions, only one of which is coated with avidin to        form binding domains.    -   7-Spot-1: Plate type D from Example 1 having 7 smaller fluid        containment regions, three of which are coated with avidin to        form a binding domain.    -   7-Spot-3: Plate type D from Example 1 having 7 smaller fluid        containment egions, only one of which is coated with avidin to        form binding domains.

After standard blocking and washing procedures, a serial dilution oftag-IgG-biotin was assayed in 50 microliter volumes with 2 hourincubation time using intermittent shaking. The plates were read with a2.5-4.5 volt scan for 5 seconds. FIG. 16 shows a log-log plot of theuncorrected data. Surprisingly, the detection limits are actuallysignificantly better for the multi-array format than for the standardformat. The relative detection limits (relative to the standard 96plate) calculated for each plate type are: standard 96 (1.0), 4-spot-1(4.2), 4-spot-3 (1.4), 7-spot-1 (4.4), 7-spot-3 (2.1). This is expectedif most of the tag is captured at the working electrode spot. As thespot gets smaller, the total specific light emitted should stayconstant, while the background signal decreases with area. On averagefor all calibrators above the detection limit, the signal for 1 of the 4spots spotted is 2.7 times as high as the average signal when 3 spotsare spotted. This indicates that most (˜90%) of the tagged molecules arebeing captured on the single spot. This example demonstrates that assaysin MDMW plates having small assay domains can have the same or betterperformance as assays in conventional single domain plates.

Example 7 Multi-Analyte Immunoassay of MDMW Plates

Sandwich immunoassays for four different cytokines—interleukin 1β(IL-1β), interleukin 6 (IL-6), interferon γ (IFN-γ) and tumor necrosisfactor α (TNF-α)—were carried out simultaneously in the wells of platesmanufactured according to the design and procedure described for Plate Cin Example 1. Four capture antibodies (each selective for one of theanalytes of interest) were patterned into distinct assay domains bymicrodispensing solutions of the antibodies on the fluid containmentregions within each well (one antibody per region) and allowing theantibodies to adsorb to the surface of the working electrode. Solutions(0.25 uL) containing the antibody (at a concentration of 32 ug/mL forIL-1β and TNF-α or 64 ug/mL for IL-6 and IFN-γ) and 0.1% BSA inphosphate buffered saline were dispensed onto the fluid containmentregions using a solenoid valve controlled microdispensor (BiodotDispensor, Cartesian Technologies) and allowed to evaporate to dryness.The volume of the antibodies was sufficient to spread over all of theexposed electrode surface within a fluid containment region but wassmall enough so that the fluid did not spread past the boundary formedby the dielectric layer. After drying the antibody solution on theworking electrode, the plate tops were attached and the excess unboundantibody was removed (and uncoated surfaces blocked) by filling thewells with a solution containing 5% (w/v) bovine serum albumin (BSA) inphosphate buffered saline (PBS). The plates were incubated with theblocking solution overnight at 4° C. and then washed with PBS.

The assays were carried out by the steps of i) adding 0.02 mL of thesample to the well and incubating for 1 hour on a plate shaker; ii)washing the wells with PBS; iii) adding 0.02 mL of a solution containing2,000 ng/mL each of four detection antibodies (labeled with NHS ester 1)against the four analytes of interest and incubating for 1 hour on aplate shaker; iv) washing with PBS; v) introducing 0.1 mL of a solutioncontaining tripropylamine in phosphate buffer (ORIGEN Assay Buffer, IGENInternational) and vi) measuring ECL. FIGS. 17A-17D demonstrate thateach of the analytes of interest can be independently measured in asingle sample in a single well of a multi-well assay plate. The figuresshow ECL emitted from each assay domain as a function of theconcentration of each analyte. The introduction of a specific analyteled to a linear increase in ECL with analyte concentration (relative tothe background signal measured in the absence of any analyte) at assaydomains having capture antibodies directed against that analyte, but didnot affect the ECL at assay domains having antibodies directed againstthe other analytes. FIG. 18 shows a CCD image of the ECL emitted from asector of wells used to assay solutions containing mixtures of the fouranalytes. The highlighted well is annotated to show the arrangement ofthe four assay domains. That specific well was used to assay a samplehaving 250 pg/mL each of IL-1β and TNF-α and 8 pg/mL each of IL-6 andIFN-γ.

Example 8 Multi-Plex Assay for Total EGF Receptor andAuto-Phosphorylated EGF Receptor

This example shows an ECL assay that measures in one well of a MDMWPlate total (phosphorylated and non-phosphorylated) EGF Receptor(T-EGFR) and phosphorylated EGF Receptor (P-EGFR).

Preparation of Lysates for Multi-Plex:

-   -   1. A-431 cells were cultured in 150 mm tissue culture dishes and        serum starved overnight (DMEM supplemented with 1%        Penicillin-Streptomycin and 1% Sodium Pyruvate).    -   2. Following two rinses with serum-free media, one dish was        stimulated with 200 nM EGF in serum-free media for 15 minutes at        room temperature. The unstimulated plate was given serum-free        media only.    -   3. The cells were rinsed with two volumes of PBS.    -   4. 2 mls of a modified RIPA buffer (fresh sodium orthovanadate        added the morning of the assay) was added to the dishes. RIPA        buffer included: 1 mM neat sodium orthovanadate, 150 mM NaCl, 50        mM Tris, 6 mM Deoxycholate, 0.5% NP40, in water with a fresh        protease inhibitor tablet, 1 tablet per 10 mL buffer). Cells        were incubated with the RIPA for 10 minutes on ice.    -   5. Supernatant was collected and quantitated using the Pierce        BCA Protein Assay.

Protocol for Multiplex Assay:

-   -   1. Biotin-labeled antibodies for T-EGFR (specific for the        cytoplasmic domain of EGFR) and P-EGFR (anti-phosphotyrosine)        were prebound (1 hour) with one equivalent of avidin and        deposited by microdispensing (one antibody per region, 0.5 pmol        per region in 0.25 uL) onto two of the four fluid containment        regions in each well of a MDMW Plate (Plate C of Example 1). The        two remaining fluid containment regions were used as controls        for non-specific binding and cross-reactions. One region was        coated with Avidin only. The other was left bare but eventually        blocked with BSA.    -   2. The antibodies were allowed to dry. The wells were then        blocked for one hour at room temperature with 200 μl per well of        5% BSA in water.    -   3. The plates were washed four times with dH₂O.    -   4. 50 μg/well of lysate was added to each well of the 96 well        plates and shaken intermittently for one hour.    -   5. The plates were washed four times with dH₂O.    -   6. The Sulfo-Tag™-labeled α-EGFR antibody (50 uL of 33 nM) was        added and the binding reaction allowed to proceed for 1 hour at        room temperature with shaking. The plates were washed four times        with dH₂O.    -   7. 100 μl per well of 100 mM TPA with 400 mM gly-gly assay        buffer was added just prior to ECL analysis.    -   8. The plates were analyzed using ECL detection.

The table below compares the ECL signals measured from the T-EGFR andP-EGFR assays for lysates from stimulated and unstimulated cells. Asexpected, over the time course of the experiment, the levels of T-EGFRdo not change considerably on stimulation, however, a large increase inP-EGFR was observed.

Analyte Sample T-EGFR P-EGFR Unstimulated 24,200 57 Stimulated 23,545122

Example 9 Multi-Plex Assay for Detection of Autophosphorylated andNonphosphorylated EGF Receptor

This example shows an ECL assay that measures both nonphosphorylated EGFreceptor and EGF receptor that is phosphorylated at tyrosine 1173 in asingle well of a MDMW Plate.

A-431 cell lysates were prepared as described in Example 8, except thatseparate dishes of cells were stimulated with 0.2 nM, 5 nM and 200 nMEGF.

Protocol for Multiplex Assay:

-   -   1. Antibodies specific for EGF receptor that is        autophosphorylated at tyrosine 1173 (pY1173) and antibodies        specific for EGF receptor that is not phosphorylated at tyrosine        1173 (Y1173) were deposited by microdispensing (one antibody per        region, 0.2 pmol per region in 0.25 uL) and passively adsorbed        onto two of the four fluid containment regions in each well of a        MDMW Plate (Plate C of Example 1). The two remaining fluid        containment regions were used as controls for non-specific        binding and cross-reactions; these regions were left bare but        eventually blocked with BSA.    -   2. The antibodies were allowed to dry overnight. The wells were        then blocked for one hour at room temperature with 200 μl per        well of 5% BSA in water.    -   3. The plates were washed with PBS.    -   4. 5 μg/well of lysate was added to each well of the 96 well        plates and the plates were shaken intermittently for three        hours.    -   5. The plates were washed with PBS.    -   6. A Sulfo-TAG label labeled α-EGFR antibody directed against        the extracellular domain of the receptor (50 uL of a 33 nM        solution) was added and the binding reaction allowed to proceed        for 1 hour at room temperature with shaking. The plates were        washed four cycles with PBS.    -   7. 150 μl per well of 100 mM TPA with 400 mM gly-gly assay        buffer was added just prior to ECL analysis.    -   8. The plates were analyzed using ECL detection in a Sector HTS™        plate reader (Meso Scale Discovery).

FIGS. 19A-D demonstrate that the amount of autophosphorylation of thetyrosine at position 1173 on the EGF receptor can be controlled andquantified in a single well of a multi-well assay plate. The schematicin FIG. 19A shows placement of the pY1173 and Y1173 antibodies on twodiagonally opposed fluid containment regions in the same well. The EGFreceptor contained in A-431 cell lysates binds to the appropriatesurface immobilized antibody. Specifically, only the phosphorylatedtyrosine at position 1173 binds the pY1173 antibody, and only thenonphosphorylated tyrosine at position 1173 binds to the Y1173 antibody.Competition for binding of the receptor to more than one assay domain ina single well is circumvented in this format. The reporter α-EGFRantibody binds the extracellular domain of both receptors. The CCDimages in FIGS. 19B-D show ECL emitted from each assay domain as afunction of increasing EGF concentration. No detectableautophosphorylation of tyrosine 1173 was observed at 0.2 nM EGF,approximately 50% of the receptor was phosphorylated at 5 nM EGF, andapproximately 90% was phosphorylated at 200 nM EGF.

Example 10 Measurement of Tyrosine Kinase and Serine/Threonine KinaseActivities in a Well of a MDMW Plate

This example used an MDMW plate adapted for ECL measurements and having4 fluid containment regions on the working electrode surface exposed ineach well (Plate C of Example 1). Each of the four fluid containmentregions received 250 nL of one of the four following solutions: (i) 0.5mg/ml Poly-Glu:Tyr (4:1) (PGT) in PBS buffer with 0.015% Triton; (ii)0.2 mg/ml Myelin Basic Protein (MBP) in PBS buffer with 0.015% Triton;(iii) 0.3 mg/ml Streptavidin in PBS buffer with 0.015% Triton; (iv) 0.3mg/ml BSA solution in PBS with 0.15% Triton. The plate was then driedfor 1-1.5 hours at ambient conditions, vigorously washed with PBScontaining 0.1% Triton, washed with water and blocked in a 5% BSAsolution for at least 2 hours at room temperature. The washing includeda bottom wash using a 96-well Plate Washer from Biotech that allows thecreation of a constant flow of wash solution in the well and was veryefficient for washing out an excess of peptides/proteins from theelectrode surface. The washing with the Triton-containing solution wasfollowed by 3x washes to remove traces of Triton. After blocking, theplate was washed again to remove blocking agent prior to use.

For phosphorylation of PGT (tyrosine kinase assay), 0.05 mU/μl of c-SRCwas used; for phosphorylation of MBP (threonine kinase), 2 nM of ERK-2was used. The capture efficiency of the Streptavidin-coated domain wasdetermined by measuring the binding of bovine IgG labeled with biotinand a sulfonated form of Ru(bpy)3 (Sulfo-TAG™ label by Meso ScaleDiscovery).

Each spot (PGT, MBP, Streptavidin and BSA) was exposed to a solution ofunlabeled primary antibodies directed against phosphotyrosine andphosphorylated MBP and labeled secondary antibodies. After incubatingthe plates to allow the enzyme and binding reactions to proceed, aTPA-containing buffer was added and the plates were analyzed by ECL (nowash was required). Each point includes an average of 12 measurementswith CV's of 7-10%. Table A below summarizes the results obtained fromthis experiment.

TABLE A No Enzyme/ Analyte blgG* SRC- ERK2- Domain No blgG* Only onlyonly SA 272 5,447 324 309 PGT 990 953 17,223 1,153 MBP 1,241 1,354 1,23732,810 BSA 138 134 168 209

The bold faced values are specific signals; the other numbers are ECLdue to non-specific interactions.

The PGT and MBP domains only showed high signal in the presence of thetyrosine kinase SRC and Threonine kinase (ERK2), respectively. Titrationcurves of the activity of both kinases (SRC and ERK) exhibited nearlylinear response on corresponding domains, The Streptavidin domain gave agood signal in the presence of the biotinylated analyte and did not actas a substrate for the kinases. This result demonstrates the utility ofincluding a binding domain, e.g., for capturing (and, optionally,purifying) kinases to be tested from crude samples. The BSA spot did notprovide a significant signal in the presence of the analyte/enzymes andshows that the blocking agent did not show non-specific reactions withthe assay reagents.

INCORPORATION OF REFERENCES

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theclaims. Various publications are cited herein, the disclosures of whichare incorporated by reference in their entireties.

1. An assay module for conducting luminescence assays comprising aplurality of assay domains, said assay domains comprising a first assaydomain having a first reagent and a second assay domain having a secondreagent, wherein said first assay domain is capable of generatingluminescence at least 10 times brighter than said second assay domainand said first assay domain and said second assay domains are notadjacent and/or are separated by at least one other assay domain toreduce luminescence emitted from said first assay domain frominterfering with luminescence emitted from said second assay domain. 2.The assay module of claim 1, wherein said first assay domain comprisesat least 10 times capture reagent than said second assay domain.
 3. Theassay module of claim 1, wherein said first assay domain is capable ofgenerating luminescence at least 100 times brighter than said secondassay domain.
 4. The assay module of claim 1, wherein said first assaydomain is capable of generating luminescence at least 1,000 timesbrighter than said second assay domain.
 5. The assay module of claim 1,wherein said second assay domain has one or more properties thatdiminish emitted luminescence.
 6. The assay module of claim 5, whereinsaid one or more properties include a capture reagent that diminishesemitted luminescence and/or a target analyte that diminishes emittedluminescence.
 7. The assay module of claim 1, wherein said first assaydomain is separated from said second assay domain by at least one otherassay domain.
 8. The assay module of claim 1, wherein said first assaydomain and said second assay domain are separately addressable.
 9. Theassay module of claim 1, wherein said assay module is a multi-well platecomprising a plurality of wells, each of said wells having a copy ofsaid plurality of assay domains.
 10. An assay module comprising aplurality of assay domains for measuring a panel of analytes, said panelcomprising a plurality of analytes that are members of an analytecategory selected from cytokines and/or their receptors; growth factorsand/or their receptors; second messengers; drugs of abuse; therapeuticdrugs; auto-antibodies; allergen specific antibodies; tumor markers;cardiac markers; markers associated with hemostasis; markers of acuteviral hepatitis infection; markers of Alzheimers Disease; markers ofosteoporosis; markers of fertility; markers of congestive heart failure;markers of thyroid disorders; markers of prostrate cancer; nucleic acidarrays for measuring mRNA levels of mRNA coding for cytokines, growthfactors, components of the apoptosis pathway, expression of the P450enzymes or expression of tumor related genes; nucleic acid arrays forgenotyping individuals, pathogens, or tumor cells.
 11. An assay moduleof claim 10, wherein said assay domains comprise: (i) analytes oranalogs of analytes; (ii) binding reagents that bind analytes; and/or(iii) substrates for catalytic activity of analytes.
 12. A method ofperforming an assay for a plurality of analytes using an assay module ofclaim 10, said method comprising: (a) contacting a sample with aplurality of assay domains within said assay module; and (b) measuringsaid analytes at said assay domains.
 13. A method for detecting orcorrecting for a source of assay error in an assay for an analytecomprising: (a) introducing a sample comprising analyte into an assaymodule comprising (i) a first assay domain comprising a first reagentcapable of binding said analyte; and (ii) a second assay domaincomprising said first reagent; wherein said first assay domain and saidsecond binding domain having different properties, said differentproperties selected from the group consisting of surface area, reagentconcentration, heat stability, pH stability, hydrophobic or hydrophilicproperties, co-reactants, epitope specificity, binding kinetics,non-specific binding properties, binding constants, and/orthermodynamics; and (b) comparing the binding of the analyte to thefirst and second assay domains so as to detect or correct for sources ofassay error.
 14. An assay module for conducting an assay on a samplecomprising an analyte and at least one interferent, said modulecomprising: (a) an assay domain capable of binding said analyte; and (b)a sequestering domain capable of binding said interferent.
 15. The assaymodule of claim 14, wherein said module is adapted to measure saidinterferent bound to said sequestering domain.
 16. A method formeasuring an analyte in the presence of an interferent comprising: (a)introducing the sample into the assay module of claim 14; and (b)detecting the binding of said analyte to said assay domain.
 17. An assaymodule comprising a plurality of assay domains, wherein said pluralityof assay domains comprises one or more assay domains comprising one ormore binding reagents and further comprising a first control domaincomprising pre-bound label, a second control domain providing a definedreaction and a third control domain consisting essentially of blockingagent.
 18. The assay module of claim 17, wherein said first controldomain controls for one or more of the following: (i) chemicalinterference with ECL generation; (ii) optical interference with lighttransmission; and/or (iii) module variation.
 19. The assay module ofclaim 17, wherein said second control domain controls for one or more ofthe following: (i) non-specific binding or biochemical activity; (ii)chemical interference with ECL generation; (iii) optical interferencewith light transmission; (iv) pipetting errors; (v) timing errors; (vi)variations in mixing; (vii) variations in temperature; and (viii) assaymodule variation.
 20. The assay module of claim 17, wherein said thirdcontrol domain controls for non-specific binding.
 21. An assay modulecomprising one or more assay domains comprising assay reagents and oneor more sequestrating domains comprising sequestration agents forsequestering interfering species.
 22. The assay module of claim 21,wherein said one or more sequestrating domains sequest one or more ofthe following interfering species: biotin; anti-Streptavidin;hemoglobin; bilirubin; lipid; high and low albumin; HAMA;anti-ruthenium; high Rheumatoid Factor; DLIF's for digoxin or digitoxintype molecules; drugs/co meds; cross reactive analytes; and combinationsthereof.
 23. An assay module comprising one or more assay domainscomprising assay reagents and one or more sequestrating domainscomprising sequestration agents capable of sequestering a labeledreagent.